Magnetic resonance studies of trypsin

Kang, Shyue-yue

doi:10.14288/1.0061092

UBC Theses and Dissertations

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UBC Theses and Dissertations

Magnetic resonance studies of trypsin Kang, Shyue-yue 1974

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Title	Magnetic resonance studies of trypsin
Creator	Kang, Shyue-yue
Publisher	University of British Columbia
Date Issued	1974
Description	A great advance in the understanding of the mechanism for enzymatic reactions on a molecular basis has resulted from knowledge of the three dimensional structure of several enzymes from x-ray diffraction methods. It is not possible, however, to determine the enzyme mechanism only by knowing its three dimensional structure. The dynamic aspect of the enzymatic reaction is required to understand its mechanism. Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) are physical methods which contain both structural and dynamic information. This thesis presents studies of the interactions between the macromolecule, trypsin, and the small molecules, substrate like inhibitors, or ions, Ca⁺⁺, Mn⁺⁺ by magnetic resonance. The theories of nuclear magnetic relaxation in the presence of chemical exchange, relaxation mechanisms, and the methods of measurement of relaxation are presented in Chapter 1. Here are discussed the equations relating measured relaxation times to chemical exchange rates, chemical shifts and relaxation times of nuclei on small molecules in dynamic chemical exchange to a macromolecular site. Different exchange limits are discussed and means for distinguishing various limits are provided. In order to understand the effect of divalent ions (Ca⁺⁺, Mn⁺⁺, etc.) on the properties of trypsin, a study of Mn⁺⁺ binding to trypsin is described in Chapter II. Mn⁺⁺ was chosen as a model for Ca⁺⁺ binding, since Mn⁺⁺ is paramagnetic. Although all previous attempts to use NMR to interpret Mn⁺⁺ binding were based on use of "enhancement factors", I found that a more straightforward and clearer approach was to use the NMR relaxation times directly. The existing theory for effect of chemical exchange on NMR T-₁'s was extended to the case of three distinct chemical sites with all possible mutual inter-conversions, and applied the result to the binding of water to free Mn⁺⁺ and Mn⁺⁺ :enzyme complex. An exact treatment of the correction for the internal rotation of water at the Mn⁺⁺ binding site is also presented. The main conclusions were that Mn⁺⁺ binds strongly only on active trypsin and at just one site, and that the water bound to Mn⁺⁺ at that site can rotate rather freely, suggesting that the site must lie in an open region of the tertiary structure. These facts are most consistent with the binding of Mn⁺⁺at Asp 71 and Asp 153 (or Glu 77), where it has been suspected (but not shown) that Ca⁺⁺ may act to hold two loops of the enzyme together. The next two chapters are devoted to the study of the active site of trypsin by UV and NMR. This basic approach was to choose a homologous series of substrate-like inhibitors of trypsin, and study their binding to trypsin both by steady- state (uv) kinetics and also by NMR relaxation time measurements. This work would provide for the first time a direct comparison between the strength of binding (as measured by the binding constant from uv data) and the rigidity with which the inhibitor was bound to the enzyme (from NMR data). Any correlation, or lack of it, between these two parameters should provide more insight into theories of enzyme action. For rigorous NMR analysis, it was desirable to have inhibitors with - OCH₃ groups, to obtain a single, sharp NMR signal well-separated from other parts of the NMR spectrum. This requirement entailed the synthesis of a number of inhibitors, and in most cases, a given synthesis was not in the literature and had to be devised individually. This is described in the experimental section of Chapter III. In order to locate the binding sites of each inhibitor, and to obtain an accurate dissociation constant for each inhibitor, uv steady-state trypsin assays using D,L-BAPA as substrate were carried out. As a result, five of the interesting inhibitors gave Dixon plots with intersections below the x-axis, a result which cannot be explained by previous trypsin inhibitor work. The difficulty was eventually resolved by taking into account the interaction between the D-BAPA and my inhibitors. Although apparently a complication, the algebraic consequences showed that my seven trypsin inhibitors could be classified according to whether their binding was competitive, repulsive, non-competitive, or cooperative with the binding of D-BAPA. This then gave a rather complete picture of the inhibitor binding. The data definitely showed the presence of at least one secondary binding site, which is consistent with a number of unpublished X-ray results, and the secondary binding site exhibits some cooperative effect toward binding of a substrate analog. This had been observed on TAME substrate activation at high concentration. Chapter IV presents the measurements of the bound relaxation time of each of the inhibitors on trypsin by selective pulsed high resolution NMR (The measurement was made on the single sharp line of the methyl protons of the inhibitors). A special-purpose pulse unit is described briefly and the advantages and limitation of selective determination of relaxation time on high resolution NMR was also discussed. The relaxation time for the rigidly bound inhibitor was calculated and a correction for the effect of internal rotation of methyl group was made. The results strongly suggest that for the inhibitors of the same category (from uv), correlation between strength of binding and rigidity of binding can be demonstrated. The resultant implications for theories of enzyme catalysis was also discussed. In the last chapter, an ESR "spin label" to the active site serine of trypsin, with the intent of using the effect of this spin label on the NMR lineshape of my inhibitors has been used as a "ruler" to determine the distance from the active site to the secondary binding sites. Hopefully, the interaction between the "spin label" and Mn⁺⁺ on the enzyme is strong enough so that the distance, between them can also be estimated. The conformational change of the active site region with different purtubations (pH, Ca⁺⁺, inhibitors, etc) was also expected to be monitored through the changes of the ESR signal of the attached spin label. In order to ensure the protection of the spin labelled trypsin from autoproteolysis during the NMR experiments, the active trypsin-free, spin labelled trypsin was prepared successfully by a new process using soybean trypsin inhibitor. From the NMR measurements, it can be estimated that the distances between the spin label and the secondary binding sites are all around 9 to 10 Å. With the help of X-ray data, the location of the secondary sites may be speculated upon and the consequence evaluated. Due to the long distance between the spin label and the Mn⁺⁺ ion binding site, the interaction between these two paramagnetic species can not be observed. In addition, the ESR signal of the ser-195 spin label was not sensitive to the minor conformational changes induced by the various perturbations added.
Genre	Thesis/Dissertation
Type	Text
Language	eng
Date Available	2010-01-28
Provider	Vancouver : University of British Columbia Library
Rights	For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
DOI	10.14288/1.0061092
URI	http://hdl.handle.net/2429/19224
Degree	Doctor of Philosophy - PhD
Program	Chemistry
Affiliation	Science, Faculty of Chemistry, Department of
Degree Grantor	University of British Columbia
Campus	UBCV
Scholarly Level	Graduate
AggregatedSourceRepository	DSpace

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C ! MAGNETIC RESONANCE STUDIES OF TRYPSIN by . SHYUE.YUE KANG B.Sc. ( H o n s . ) , N a t i o n a l Taiwan U n i v e r s i t y , C h i n a , 1967. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f C h e m i s t r y We a c c e p t t h i s t h e s i s as comforming t o t h e r e q u i r e d s t a n d a r d - — THE UNIVERSITY OF BRITISH COLUMBIA 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date TktST, - i i -A b s t r a c t A g r e a t advance i n t h e u n d e r s t a n d i n g o f t h e mechanism f o r e n z y m a t i c r e a c t i o n s on a m o l e c u l a r b a s i s has r e s u l t e d from knowledge o f t h e t h r e e d i m e n s i o n a l s t r u c t u r e o f s e v e r a l enzymes f r o m x - r a y d i f f r a c t i o n methods. I t i s not p o s s i b l e , however, t o d e t e r m i n e the enzyme mechanism o n l y by knowing i t s t h r e e d i m e n s i o n a l s t r u c t u r e . The dynamic a s p e c t o f t h e e n z y m a t i c r e a c t i o n i s r e q u i r e d t o u n d e r s t a n d i t s mechanism. N u c l e a r m a g n e t i c r e s o n a n c e (NMR) and e l e c t r o n s p i n r e s o n a n c e (ESR) a r e p h y s i c a l methods which c o n t a i n b o t h s t r u c t u r a l and dynamic i n f o r m a t i o n . T h i s t h e s i s p r e s e n t s s t u d i e s o f t h e i n t e r a c t i o n s between t h e m a c r o m o l e c u l e , t r y p s i n , and the s m a l l m o l e c u l e s , s u b s t r a t e l i k e i n h i b i t o r s , o r i o n s , C a + + , M n + + by m a g n e t i c r e s o n a n c e . The t h e o r i e s o f n u c l e a r m a g n e t i c r e l a x a t i o n i n t h e p r e s e n c e o f c h e m i c a l exchange, r e l a x a t i o n mechanisms, and t h e methods o f measurement o f r e l a x a t i o n a r e p r e s e n t e d i n C h a p t e r 1. Here a r e d i s c u s s e d t h e e q u a t i o n s r e l a t i n g measured r e l a x a t i o n t i m e s t o c h e m i c a l exchange r a t e s , c h e m i c a l s h i f t s and r e l a x a t i o n t i m e s o f n u c l e i on s m a l l m o l e c u l e s i n dynamic c h e m i c a l exchange t o a m a c r o m o l e c u l a r s i t e . D i f f e r e n t exchange l i m i t s a r e d i s c u s s e d and means f o r d i s t i n g u i s h i n g v a r i o u s l i m i t s a r e p r o v i d e d . - i i i -In o r d e r t o u n d e r s t a n d t h e e f f e c t o f d i v a l e n t i o n s ( C a + + , Mn , e t c . ) on t h e p r o p e r t i e s o f t r y p s i n , a s t u d y o f Mn b i n d i n g t o t r y p s i n i s d e s c r i b e d i n C h a p t e r I I . Mn was chose n as a model f o r Ca b i n d i n g , s i n c e Mn i s p a r a m a g n e t i c . A l t h o u g h a l l p r e v i o u s a t t e m p t s t o use NMR t o i n t e r p r e t M n + + b i n d i n g were based on use o f "enhancement f a c t o r s " , I f o u n d t h a t a more s t r a i g h t f o r w a r d and c l e a r e r a p p r o a c h was t o use t h e NMR r e l a x a t i o n t i m e s d i r e c t l y . The e x i s t i n g t h e o r y f o r e f f e c t o f c h e m i c a l exchange on NMR T-j's was ex t e n d e d t o t h e c a s e o f t h r e e d i s t i n c t c h e m i c a l s i t e s w i t h a l l p o s s i b l e mutual i n t e r -c o n v e r s i o n s , and a p p l i e d t h e r e s u l t t o t h e b i n d i n g o f w a t e r t o f r e e Mn and Mn :enzyme complex. An e x a c t t r e a t m e n t o f t h e c o r r e c t i o n f o r t h e i n t e r n a l r o t a t i o n o f w a t e r a t t h e Mn b i n d i n g s i t e i s a l s o p r e s e n t e d . The main c o n c l u s i o n s were t h a t M n + + b i n d s s t r o n g l y o n l y on a c t i v e t r y p s i n and a t j u s t one s i t e , and t h a t t h e water bound t o Mn a t t h a t s i t e can r o t a t e r a t h e r f r e e l y , s u g g e s t i n g t h a t t h e s i t e must l i e i n an open r e g i o n o f t h e t e r t i a r y s t r u c t u r e . These f a c t s a r e most c o n s i s t e n t w i t h t h e b i n d i n g o f M n + + a t Asp 71 and Asp 153 ( o r G l u 7 7 ) , where i t has been s u s p e c t e d ( b u t n o t shown) t h a t C a + + may a c t t o h o l d two l o o p s o f t h e enzyme t o g e t h e r . The n e x t two c h a p t e r s a r e d e v o t e d t o t h e s t u d y o f t h e a c t i v e s i t e o f t r y p s i n by UV and NMR. T h i s b a s i c a p p r o a c h was t o choos e a homologous s e r i e s o f s u b s t r a t e - l i k e i n h i b i t o r s o f t r y p s i n , and s t u d y t h e i r b i n d i n g t o t r y p s i n b o t h by s t e a d y -- i v -s t a t e (uv) k i n e t i c s and a l s o by NMR r e l a x a t i o n t i m e measurements. T h i s work would p r o v i d e f o r t h e f i r s t t i me a d i r e c t c o m p a r i s o n between t h e s t r e n g t h o f b i n d i n g (as measured by t h e b i n d i n g c o n s t a n t f r o m uv d a t a ) and t h e r i g i d i t y w i t h w hich t h e i n h i b i t o r was bound t o t h e enzyme ( f r o m NMR d a t a ) . Any c o r r e l a t i o n , o r l a c k o f i t , between t h e s e two p a r a m e t e r s s h o u l d p r o v i d e more i n s i g h t i n t o t h e o r i e s o f enzyme a c t i o n . F o r r i g o r o u s NMR a n a l y s i s , i t was d e s i r a b l e t o have i n h i b i t o r s w i t h - OCHg g r o u p s , t o o b t a i n a s i n g l e , s h a r p NMR s i g n a l w e l l - s e p a r a t e d f r o m o t h e r p a r t s o f t h e NMR s p e c t r u m . T h i s r e q u i r e m e n t e n t a i l e d t h e s y n t h e s i s o f a number o f i n h i b i t o r s , and i n most c a s e s , a g i v e n s y n t h e s i s was n o t i n t h e l i t e r a t u r e and had t o be d e v i s e d i n d i v i d u a l l y . T h i s i s d e s c r i b e d i n t h e e x p e r i m e n t a l s e c t i o n o f C h a p t e r I I I . In o r d e r t o l o c a t e t h e b i n d i n g s i t e s o f each i n h i b i t o r , and t o o b t a i n an a c c u r a t e d i s s o c i a t i o n c o n s t a n t f o r each i n h i b i t o r , uv s t e a d y - s t a t e t r y p s i n a s s a y s u s i n g D.L-BAPA as s u b s t r a t e were c a r r i e d o u t . As a r e s u l t , f i v e o f t h e i n t e r e s t i n g i n h i b i t o r s g a v e Dixon p l o t s w i t h i n t e r s e c t i o n s below t h e x - a x i s , a r e s u l t w h i c h c a n n o t be e x p l a i n e d by p r e v i o u s t r y p s i n i n h i b i t o r work. The d i f f i c u l t y was e v e n t u a l l y r e s o l v e d by t a k i n g i n t o a c c o u n t t h e i n t e r a c t i o n between the D-BAPA and my i n h i b i t o r s . A l t h o u g h a p p a r e n t l y a c o m p l i c a t i o n , the a l g e b r a i c c o nsequences showed t h a t my s e v e n t r y p s i n i n h i b i t o r s c o u l d be c l a s s i f i e d a c c o r d i n g t o w h e t her t h e i r b i n d i n g was c o m p e t i t i v e , r e p u l s i v e , n o n - c o m p e t i t i v e , o r c o o p e r a t i v e w i t h the b i n d i n g o f D-BAPA. T h i s t h e n gave a r a t h e r c o m p l e t e p i c t u r e o f t h e i n h i b i t o r b i n d i n g . The d a t a d e f i n i t e l y showed the - V -p r e s e n c e o f a t l e a s t one s e c o n d a r y b i n d i n g s i t e , w h ich i s c o n s i s t e n t w i t h a number o f u n p u b l i s h e d X - r a y r e s u l t s , and t h e s e c o n d a r y b i n d i n g s i t e e x h i b i t s some c o o p e r a t i v e e f f e c t toward b i n d i n g o f a s u b s t r a t e a n a l o g . T h i s had been o b s e r v e d on TAME s u b s t r a t e a c t i v a t i o n a t h i g h c o n c e n t r a t i o n . C h a p t e r IV p r e s e n t s t h e measurements o f t h e bound r e l a x a t i o n t i m e o f e a c h o f t h e i n h i b i t o r s on t r y p s i n by s e l e c t i v e p u l s e d h i g h r e s o l u t i o n NMR (The measurement was made on t h e s i n g l e s h a r p l i n e o f t h e m e t h y l p r o t o n s o f t h e i n h i b i t o r s ) . A s p e c i a l -p u r p o s e p u l s e u n i t i s d e s c r i b e d b r i e f l y and t h e a d v a n t a g e s and l i m i t a t i o n o f s e l e c t i v e d e t e r m i n a t i o n o f r e l a x a t i o n t i m e on h i g h r e s o l u t i o n NMR was a l s o d i s c u s s e d . The r e l a x a t i o n t i m e f o r the r i g i d l y bound i n h i b i t o r was c a l c u l a t e d and a c o r r e c t i o n f o r the e f f e c t o f i n t e r n a l r o t a t i o n o f methyl group v/as made. The r e s u l t s s t r o n g l y s u g g e s t t h a t f o r t h e i n h i b i t o r s o f t h e same c a t e g o r y ( f r o m u v ) , c o r r e l a t i o n between s t r e n g t h o f b i n d i n g and r i g i d i t y o f b i n d i n g can be d e m o n s t r a t e d . The r e s u l t a n t i m p l i c a t i o n s f o r t h e o r i e s o f enzyme c a t a l y s i s was a l s o d i s c u s s e d . In t h e l a s t c h a p t e r , an ESR " s p i n l a b e l " t o t h e a c t i v e s i t e s e r i n e o f t r y p s i n , w i t h t h e i n t e n t o f u s i n g t h e e f f e c t o f has been used t h i s s p i n l a b e l on t h e NMR l i n e s h a p e o f my i n h i b i t o r s A a s a " r u l e r " t o d e t e r m i n e t h e d i s t a n c e f r o m t h e a c t i v e s i t e t o t h e s e c o n d a r y b i n d i n g s i t e s . H o p e f u l l y , t h e i n t e r a c t i o n between the " s p i n l a b e l " and M n + + on t h e enzyme i s s t r o n g enough so t h a t t h e d i s t a n c e , between them can a l s o be e s t i m a t e d . The c o n f o r m a t i o n a l change o f - v i -t h e a c t i v e s i t e r e g i o n w i t h d i f f e r e n t p u r t u b a t i o n s (pH, C a + + , i n h i b i t o r s , e t c ) was a l s o e x p e c t e d t o be m o n i t o r e d t h r o u g h t h e changes o f t h e ESR s i g n a l o f t h e a t t a c h e d s p i n l a b e l . In o r d e r t o e n s u r e t h e p r o t e c t i o n o f the s p i n l a b e l l e d t r y p s i n from a u t o p r o t e o l y s i s d u r i n g t h e NMR e x p e r i m e n t s , t h e a c t i v e t r y p s i n - f r e e , s p i n l a b e l l e d t r y p s i n was p r e p a r e d s u c c e s s f u l l y by a new p r o c e s s u s i n g soybean t r y p s i n i n h i b i t o r . From t h e NMR measurements, i t can be e s t i m a t e d t h a t t h e d i s t a n c e s between t h e s p i n l a b e l and t h e o s e c o n d a r y b i n d i n g s i t e s a r e a l l around 9 t o 10 A. With t h e h e l p o f X - r a y d a t a , t h e l o c a t i o n o f t h e s e c o n d a r y s i t e s may be upon s p e c u l a t e c j ^ a n d t h e c o n s e q u e n c e e v a l u a t e d . Due t o t h e l o n g d i s t a n c e between t h e s p i n l a b e l and t h e M n + + i o n b i n d i n g s i t e , t h e i n t e r a c t i o n between t h e s e two p a r a m a g n e t i c s p e c i e s can not be o b s e r v e d . In a d d i t i o n , t h e ESR s i g n a l o f t h e s e r - 1 9 5 s p i n l a b e l was not s e n s i t i v e t o t h e m i n o r c o n f o r m a t i o n a l changes i n d u c e d by t h e v a r i o u s p e r t u r b a t i o n s added. S u p e r v i s o r . - v i i -TABLE OF CONTENTS Page A b s t r a c t i i T a b l e o f C o n t e n t s v i i L i s t o f T a b l e s . x i L i s t o f F i g u r e s x i i i Acknowledgements x v i CHAPTER I B a s i s o f M a g n e t i c Resonance Methods f o r Stud y o f B i o l o g i c a l M a c r o m o l e c u l e s i n S o l u t i o n A. I n t r o d u c t o r y Remarks 1 B. N u c l e a r R e l a x a t i o n and Chemical Exchange ,.. 3 C. Chemical S h i f t s and Chemical Exchange 12 D. The Mechanism o f t h e R e l a x a t i o n P r o c e s s .... 12 E. Methods o f Measurement 15 1. Measurement o f s p i n - l a t t i c e r e l a x a t i o n t i m e , T-j, 15 2. Measurement o f s p i n - s p i n r e l a x a t i o n t i m e , T 2 21 3. F o u r i e r - T r a n s f o r m NMR 24 R e f e r e n c e s ( C h a p t e r I) ......... 28 CHAPTER II M a g n e t i c Resonance S t u d i e s o f Mn(II) Ions « i B i n d i n g t o T r y p s i n A. I n t r o d u c t i o n 30 B. T h e o r y 31 C. E x p e r i m e n t a l • 35 D. R e s u l t s and D i s c u s s i o n 39 - v i i i -1. S t r e n g t h and number o f M n T T - b i n d i n g s i t e s on t r y p s i n . 39 2. R o t a t i o n a l l a b i l i t y o f w a t e r a t t h e s t r o n g M n + + - b i n d i n g s i t e o f t r y p s i n 44 E. Summary 57 R e f e r e n c e s ( C h a p t e r I I ) .... 58 CHAPTER I I I S t e a d y - S t a t e I n h i b i t i o n K i n e t i c s U s i n g Racemic S u b s t r a t e : A Probe F o r C o o p e r a t i v e I n h i b i t o r B i n d i n g In T r y p s i n A. I n t r o d u c t i o n ...... 60 B. E x p e r i m e n t a l .. „.. 63 C. T h e o r y .. „ 66 D. R e s u l t s and D i s c u s s i o n 70 E. Summary 78 R e f e r e n c e s ( C h a p t e r I I I ) 80 CHAPTER IV The S t u d i e s o f R i g i d i t y o f B i n d i n g o f I n h i b i t o r s t o T r y p s i n by NMR and i t s C o r r e l a t i o n w i t h the S t r e n g t h o f B i n d i n g A. I n t r o d u c t i o n > 82 B. E x p e r i m e n t a l 84 1. I n s t r u m e n t 34 2. Method o f Measurement 87 a. c h e m i c a l s h i f t .. 87 - i x -b. r e l a x a t i o n t i m e 87 C. R e s u l t s and D i s c u s s i o n 89 D. Summary • 107 R e f e r e n c e s ( C h a p t e r IV) 108 CHAPTER V M a g n e t i c Resonance S t u d i e s o f S e r i n e -1 9 5 - S p i n - L a b e l e d T r y p s i n A. I n t r o d u c t i o n I l l B. E x p e r i m e n t a l 113 1. M a t e r i a l s 113 2. P r e l i m i n a r y p r e p a r a t i o n o f g e l f i l t r a t i o n columns 114 3. P r e p a r a t i o n o f a c t i v e t r y p s i n - f r e e , s p i n l a b e l e d t r y p s i n 114 4. Method 131 C. T h e o r y 132 D. A n a l y s i s o f Data 134 1. D e t e r m i n a t i o n o f T 2 M and y 134 2. xr i s dominated by T 136 C Y 3. J u s t i f i c a t i o n f o r the as s u m p t i o n o f f a s t exchange 137 E. R e s u l t s and D i s c u s s i o n 139 1. E s t i m a t i o n o f d i s t a n c e by m e a s u r i n g t h e i n d u c e d n u c l e a r r e l a x a t i o n 139 2. C o n f o r m a t i o n a l changes examined by ESR 151 i - X -F. Summary 153 R e f e r e n c e s ( C h a p t e r V) 154 APPENDIX A The l o n g i t u d i n a l R e l a x a t i o n Time T ^ f o r A T h r e e S i t e System 156 APPENDIX B Comments on F a s t Exchange L i m i t f o r S p i n L a b e l Enchancement o f N u c l e a r R e l a x a t i o n 161 - x i -LIST OF TABLES T a b l e T i t l e Page No. 1.1 V a r i o u s l i m i t s f o r c h e m i c a l exchange and i t s d e t e r m i n a t i o n 9 2.1 E x p e r i m e n t a l p r o t o n r e l a x a t i o n r a t e s ( s e c ~ ^ ) f o r w a t e r p r o t o n s i n the p r e s e n c e and absence o f t r y p s i n a t d i f f e r e n t c o n c e n t r a t i o n o f M n + + s o l u t i o n s 46 3.1 I n t e r p r e t a t i o n o f t h e s i g n i f i c a n c e o f the y - v a l u e s a t the i n t e r s e c t i o n o f two Dixon p l o t s c o r r e s p o n d i n g t o two d i f f e r e n t c o n c e n t r a t i o n s o f a r a c e m i c " s u b s t r a t e " 73 3.2 S t r u c t u r e s , i n h i b i t i o n c o n s t a n t s , and i n t e r a c t i o n c o e f f i c i e n t s f o r b i n d i n g o f i n h i b i t o r s t o t r y p s i n ( o u r s t u d i e s ) 74 3.3 S t r u c t u r e s , i n h i b i t i o n c o n s t a n t s , and i n t e r a c t i o n c o e f f i c i e n t s f o r b i n d i n g o f i n h i b i t o r s t o t r y p s i n . ( M a r e s - G u i a and Shaw, 1965) 76 4.1 S t r e n g t h o f b i n d i n g ( K I ) , c o o p e r a t i v i t y towards b i n d i n g o f s u b s t r a t e ( A ) , and r i g i d i t y o f b i n d i n g ( l / T g ) o f v a r i o u s i n h i b i t o r s t o t r y p s i n 102 - x i i -LIST OF TABLES ( C o n t i n u e d ) T a b l e T i t l e Page No. 5.1 E x p e r i m e n t a l p a r a m e t e r s used i n enzyme i n t r a m o l e c u l a r c a l c u l a t i o n s ( a t t e m p e r a t u r e 3 0 ° ± 1° C) . 140 5.2 E x p e r i m e n t a l p a r a m e t e r s used i n e s t i m a t i o n o f t h e e l e c t r o n - n u c l e a r d i p o l e - d i p o l e c o n t r i b u t i o n t o i n h i b i t o r p r o t o n n u c l e a r r e l a x a t i o n due t o S L - t r y p s i n a t a d i f f e r e n t t e m p e r a t u r e 140 5.3 Length o f i n h i b i t o r m o l e c u l e s and t h e p o s s i b l e l o c a t i o n o f s e c o n d a r y s i t e s 149 - x i i i -LIST OF FIGURES. F i g u r e T i t l e Page No. 1.1 Computer s i m u l a t i o n o f the t e m p e r a t u r e b e h a v i o r o f T 2 a t 60 MHz, 100 MHz and 220 MHz 10 1.2 Measurement o f T-j : Method #1 17 1.3 Measurement o f T^ : Method #2 19 1.4 Measurement o f N u c l e a r T 2 21 1.5 T r a n s i e n t . a n d F o u r i e r - T r a n s f o r m e d NMR Responses f o r an I n h i b i t o r o f T r y p s i n 25 2.1 ESR Spectrum o f 1 0 ~ 4 M/a M n + + ~ s o l u t i o n a t pH 7.1 and 21 ± 0.5° C 37 2.2 S c a t c h a r d p l o t f o r d e t e r m i n a t i o n o f s t r e n g t h and number o f b i n d i n g s i t e s f o r M n + + t o t r y p s i n 41 2.3 The p o s s i b l e s t r u c t u r e o f M n + + s t r o n g b i n d i n g s i t e on t r y p s i n 52 2.4 L o g - l o g p l o t o f l o n g i t u d i n a l r e l a x a t i o n r a t e v e r s u s m a c r o m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n time 54 3.1 Dixon p l o t f o r i n h i b i t o r o f t r y p s i n by the i n h i b i t o r s shown i n each p l o t 71 - x i v -LIST OF FIGURES ( C o n t i n u e d ) F i g u r e T i t l e Page No. 4.1 Computer s i m u l a t i o n f o r 1/T 2 v e r s u s [ E 0 ' ] / [ I 0 ] x 1 0 3 a t d i f f e r e n t h y p o t h e t i c a l c o n c e n t r a t i o n w i t h [E ] = 1 0 " 3 M/£ 90 4.2-4.2 l / L , and 1/T-, f o r the methyl p r o t o n s o f t h e i n h i b i t o r s a t d i f f e r e n t c o n c e n t r a t i o n s i n the p r e s e n c e o f c o n s t a n t amounts o f t r y p s i n , [E ] = 1 0 " 3 M/a, PD = 8.1. T emperature 30 ± 1° C 92-99 5.1 S e p e r a t i o n o f t r y p s i n - s o y b e a n t r y p s i n i n h i b i t o r (STI) complex from a m i x t u r e o f s p i n - l a b e l e d t r y p s i n , t r y p s i n - S T I complex and STI by chromatograph on column (5.0 x 80 cm) o f Sephadex G-50 > 117 5.2 S e p e r a t i o n o f a and g s p i n l a b e l e d t r y p s i n from a m i x t u r e o f a and g s p i n l a b e l e d t r y p s i n , S T I , and i n a c t i v e ( s p i n l a b e l e d ) t r y p s i n by i o n exchange chromatography on a column (2.6 x 50 cm) o f SP-Sephadex C-50 .. ng 5.3 ESR s p e c t r u m o f f r e e s p i n l a b e l , s p i n l a b e l e d a and g t r y p s i n 121 5.4 A c o m p a r i s o n o f the ESR s p e c t r a o f s p i n l a b e l e d t r y p s i n b e f o r e and a f t e r f r e e z e d r y i n g .. 123 - X V -LIST OF FIGURES ( C o n t i u n e d ) F i g u r e T i t l e Page No. 5.5 The s t a b i l i t y o f the a c t i v e t r y p s i n - f r e e , s p i n - l a b e l e d t r y p s i n examined by ESR 125 5.5 The h y d r o l y s i s o f s p i n - l a b e l e d t r y p s i n i n the p r e s e n c e o f commercial t r y p s i n 127 ++ 5.7 A. ESR s p e c t r a o f Mn i n t h e p r e s e n c e o f s p i n l a b e l e d t r y p s i n B.C.D. ESR s p e c t r a o f s p i n l a b e l e d t r y p s i n w i t h d i f f e r e n t p u r t u b a t i o n s ..... 129 5.8 T y p i c a l P r o t o n H i g h - R e s o l u t i o n Spectrum o f an i n h i b i t o r o f T r y p s i n 143 5.9 T y p i c a l e f f e c t o f S L - T r y p s i n on P r o t o n NMR s p e c t r u m o f an i n h i b i t o r 145 - xvi -ACKNOWLEDGEMENT I would like to express my sincere gratitude to my research director, Dr. A.G. Marshall, for his guidance, invaluable advice, . and constant encouragement throughout my work. Thanks are also due to Dr. B.A. Dunell for his kind permission to use the Bruker NMR pulse Spectrometer. Dr. L.D. Hall for his kind permission to use the Audio-Frequency Pulse Unit. Dr. K.A. Walsh for permitting me to work in his laboratory and for his advice in conduction of my trypsin assays. Dr. D.G. Clark for his advice on my enzyme preparation and his help on various biochemical technigues. Dr. L. WeiIer and his research group for advice and suggestion in the synethetic work. Dr. D.T. Suzuki for use of his U.V. spectrometer. Dr. P.D. Bragg for his stimulating suggestions in theory of enzyme kinetics. ' My colleagues Dr. P. Griffth, L.G. Werbelow and P.J. Morrod for their friendship and help. The members of electronic shop for their ideal service on the spectrometers. - 1 -CHAPTER I Basis of Magnetic Resonance Methods for Study of Biological MacromoTecules in Solution A. Introductory remarks Only two types of spectroscopic methods are capable of detecting individual atoms in macromolecules, x-ray diffraction in the crystalline state and magnetic resonance in the liquid state. Beth'types of information are required for unravelling the central question in biological chemistry: the relation between molecular structure and function. The exciting success of x-ray diffraction in reconstructing the three dimensional structure of enzymes is already established; A comparative latecomer - magnetic resonance, is just beginning to demonstrate i t ' s potentiality. Sources of information from NMR. 1) Chemical shift (6) - identification of the nuclei in different environments. 2) Coupling constant (J) - detail of molecular structure in conformation. 3) Area under the resonance signal-relative populations of nuclei in different environments. 4) S p i n - l a t t i c e and s p i n - s p i n r e l a x a t i o n t i m e (T^ and T 2 ) -dynamic i n f o r m a t i o n s u c h a s m o l e c u l a r m o t i o n s , r a t e o f c h e m i c a l e x c h a n g e s , r i g i d i t y o f b i n d i n g , and t h e d i s t a n c e between t h e i n t e r a c t i n g n u c l e i o r between t h e i n t e r a c t i n g e l e c t r o n s and n u c l e i . S i m i l a r i n f o r m a t i o n i s o b t a i n a b l e from ESR h y p e r f i n e s p l i t t i n g , peak a r e a , and l i n e s h a p e ; however, i t i s g e n e r a l l y n e c e s s a r y t o d e s i g n and s y n t h e s i z e a s p e c i f i c " s p i n - l a b e l " f o r b i n d i n g t o a p a r t i c u l a r m a c r o m o l e c u l a r s i t e . D i r e c t NMR s t u d i e s on m a c r o m o l e c u l e s a r e d i f f i c u l t t o c o n d u c t b e c a u s e o f t h e weakness o f t h e NMR s i g n a l and t h e c o m p l e x i t y o f t h e s p e c t r a . The i n t e r a c t i o n s between a s m a l l m o l e c u l e ( i n h i b i t o r ) and a m a c r o m o l e c u l e (enzyme) p r o v i d e s a means f o r a m p l i f y i n g t h e NMR s i g n a l by t a k i n g a d v a n t a g e o f t h e o b s e r v a b l e p e r t u r b a t i o n o f t h e s t r o n g NMR s i g n a l o f a (more c o n c e n t r a t e d ) s m a l l m o l e c u l e i n t h e p r e s e n c e o f a ( h i g h l y d i l u t e ) m a c r o m o l e c u l a r s o l u t i o n . In g e n e r a l , i t i s n o t e a s y t o d i r e c t l y d e t e c t changes i n c h e m i c a l s h i f t o r c o u p l i n g c o n s t a n t o f a m a c r o m o l e c u l a r s i t e . In t h e f o l l o w i n g s e c t i o n s , d i s c u s s i o n w i l l be c o n c e n t r a t e d on t h e i n f o r m a t i o n o b t a i n e d f r o m r e l a x a t i o n t i m e measurements (T-j and L , ) . The t h e o r y o f r e l a x a t i o n i n t h e p r e s e n c e o f c h e m i c a l exchange w i l l be r e v i e w e d . Some o f t h e l i m i t s f o r c h e m i c a l exchange w i l l be r e - e v a l u a t e d and d i s c u s s e d and f i n a l l y some s i m p l e p h y s i c a l d e s c r i p t i o n o f t h e d i f f e r e n t t e c h n i q u e s i n v o l v e d i n m e a s u r i n g T-j and T 2 a r e p r e s e n t e d . - 3 -B. N u c l e a r r e l a x a t i o n and c h e m i c a l exchange The i n t e r a c t i o n s between s m a l l m o l e c u l e s and m a c r o m o l e c u l e s a r e o u r main i n t e r e s t i n t h e s e s t u d i e s . In g e n e r a l , t h e s m a l l m o l e c u l e jumps back and f o r t h between an aqueous o r f r e e e n v i r -onment "A" and some b i n d i n g s i t e "B" o f t h e m a c r o m o l e c u l e K __L_v "A" v „ V " B " ( 1 . 1 ) •1 "A" u s u a l l y r e p r e s e n t s t h e s o l v e n t , d i m a g n e t i c e n v i r o n m e n t , e t c . and "B" u s u a l l y r e f e r s t o t h e a c t i v e s i t e o f t h e enzyme, o r t o t h e m a g n e t i c s p h e r e o f i n f l u e n c e o f p a r a m a g n e t i c i o n ; K-. i s t h e r a t e c o n s t a n t f o r t h e s m a l l m o l e c u l e t o jump t o "B" s t a t e ; and K_.| i s t h e r a t e c o n s t a n t f o r i t jumping back i n t o t h e s o l v e n t . M c C o n n e l l ^ m o d i f i e d t h e B l o c h e q u a t i o n t o i n c l u d e t h e p r o c e s s o f c h e m i c a l exchange between two m a g n e t i c a l l y d i s t i n c t s i t e s . T h i s t r e a t m e n t was e x t e n d e d t o 3 s i t e s and v a r i o u s l i m i t s 2 were e x p l o r e d by S w i f t and C o n n i c k . When one c h e m i c a l component was i n g r e a t e x c e s s (as i n enzyme k i n e t i c s ) t h e a s s u m p t i o n t h a t t h e f r a c t i o n o f t h e o b s e r v e d m o l e c u l e s a t s i t e A i s much g r e a t e r t h a n t h a t o f t h e m o l e c u l e a t s i t e B l e a d s t o t h e f o l l o w i n g f o r m f o r t h e o b s e r v e d t r a n s v e r s e r e l a x a t i o n t i m e ; 2 _ ( 1 . 2 ) fft fD rO/T 2 B + V T b ) T / T 2 B + A W B 1 s "A + J _ T ^2k T B ( 1 / T 2 B + l / t B ) 2 + A W B 2 where f ^ , f g a r e t h e f r a c t i o n s o f t h e o b s e r v e d m o l e c u l e s a t s i t e s - 4 -A and B respectively and » f g , tg = 1/K_^, AWg is the difference in chemical sh i f t between sites "A" and "B" in radians/ s e c , T 2 A , T 2 B are the transverse relaxation times for the nuclei at sites "A" and "B" respectively. The corresponding equation for spin-latt ice relaxation 3 time has been derived by Luz and Meiboom , where T^ A and T^ B are the longitudinal relaxation times for nuclei at sites "A" and "B" respectively. case A. AWg = 0 (no chemical " sh i f t " on binding) Here the chemical shi f t at s i te "B" is close or equal to that of s i te "A" . There wil l be only one observed resonance signal at frequency W.. Equation (2) can then be simplif ied to f, B + T (1.3) B T 2A + (1 .4) with f A " f l B f. = 1 (this condition wil l apply to a l l succeeding equations) 1imit (a) slow exchange, or 2B so that i _ 1 + f B T 2 T 2A T B (1 .5) - 5 -Rate o f c h e m i c a l exchange i s s l o w compared t o m a g n e t i c r e l a x a t i o n and t h e o b s e r v e d l i n e b r o a d e n i n g i s dominated by exchange r a t e o r l i f e t i m e b r o a d e n i n g . l i m i t (b) f a s t exchange ^ » o r T N « T , T B 12B T o n V 1 lB '2B so t h a t ' i - - r- + ^ - ( i . 6 ) '2 '2A 12B Rate o f c h e m i c a l exchange i s f a s t compared t o m a g n e t i c r e l a x a t i o n s and t h e o b s e r v e d l i n e b r o a d e n i n g i s do m i n a t e d by t h e t r a n s v e r s e r e l a x a t i o n t i m e a t s i t e "B". The t e m p e r a t u r e dependence o f l / T g p r o v i d e s t h e means o f d i s t i n g u i s h i n g between l i m i t (a) and ( b ) 4 . S i n c e 1 / i g i n c r e a s e s w i t h t e m p e r a t u r e a c c o r d i n g t o 1/T b = K - 1 = A e ( " E A / R T ) (1.7) where A i s a c o n s t a n t , i s t h e e n e r g y r e q u i r e d f o r t h e c h e m i c a l exchange p r o c e s s , R i s t h e gas c o n s t a n t , and T i s t e m p e r a t u r e , 1/T 2 s h o u l d i n c r e a s e l o g a r i t h m i c a l l y w i t h i n c r e a s i n g t e m p e r a t u r e i n l i m i t ( a ) . On t h e o t h e r hand, s i n c e 1 / T 2 B d e c r e a s e s w i t h i n c r e a s i n g t e m p e r a t u r e ^ , 1 / T 2 B « T C « | 0 . 8 ) - 6 -1/^ s h o u l d d e c r e a s e w i t h t e m p e r a t u r e i n l i m i t ( b ) . c a s e B. AWg > 0 ( m e a s u r a b l e c h e m i c a l " s h i f t " on b i n d i n g ) The s i t u a t i o n i s g r e a t l y c o m p l i c a t e d by i n t r o d u c i n g t h i s e x t r a f a c t o r , s i n c e e q u a t i o n (1 .2) can no l o n g e r be s i m p l i f i e d as i n c a s e A. F u r t h e r m o r e , t h e t e m p e r a t u r e c o n t r o l e x p e r i m e n t used i n c a s e A i s no l o n g e r s u f f i c i e n t t o d i s t i n g u i s h unambig-u o u s l y t h e d i f f e r e n t exchange l i m i t s . The l i n e b r o a d e n i n g c o u l d be do m i n a t e d by any o f t h r e e v a r i a b l e s , A W r , » ~ • The d i f f e r e n t l i m i t f o r c h e m i c a l b 1 2 B T B 2 exchange had been d i s c u s s e d by S w i f t and C o n n i c k . Each o f t h e l i m i t s w i l l be re-examined and a means o f d i s t i n g u i s h i n g among t h e s e l i m i t s w i l l be p r o v i d e d . F o r b r e v i t y , l e t l i m i t (a) AW R 2 » -X^- , T B T 2 B so t h a t The s e p a r a t i o n o f t h e two r e s o n a n c e l i n e s a t s i t e A and B i s much l a r g e r t h a n e i t h e r t h e exchange r a t e o r t h e t r a n s v e r s e r e l a x a t i o n t i m e s . T h e o r e t i c a l l y , t h e r e s h o u l d be two r e s o n a n c e l i n e s each a t r e s o n a n c e f r e q u e n c i e s W^ , Wg, i t s h o u l d be remem-b e r e d t h a t "A" i s i n g r e a t e x c e s s , so t h a t o n l y t h e r e s o n a n c e - 7 -will be visible. limit (b) -l—p » AW B 2 , or T 2 B TB <TT>NET - ( L I D C B It will be two broad lines partially collapsed or a single line depending of whether A W g » — or AWg<< ~ respectively. B B The line broadening in limit (a) and (b) is mainly con-trolled by exchange rate Tg. 1 2 1 1 limit (c) —w— » AWD and — < y — t g " TB ! 2 B (TJ>NET = V B A W B 2 t 1 ' 1 2 * The exchange rate is fast compared to the separation of the two resonanceslines at sites A and B. There will be one single line located between W^  and W g. ^I^^^^ is controlled by the rate of relaxation through the change in the precession frequency and is field dependent. limit (d) - - J — » AW R 2 and — » J— 2B T B B TB 1 2 B % > N E T - \ « • " ) This is the real fast exchange limit. Rate of chemical - 8 -exchange i s f a s t compared t o e i t h e r t h e r e l a x a t i o n t i m e s o r t h e d i f f e r e n c e s o f c h e m i c a l s h i f t s between s i t e s " A " and " B " . The l i n e b r o a d e n i n g i s due m a i n l y t o t h e r e l a x a t i o n p r o c e s s a t s i t e " B " . H e r e , t h e t e m p e r a t u r e b e h a v i o r s o f t h e " f a s t " exchange l i m i t ( c ) and (d) a r e q u a l i t a t i v e l y s i m i l a r , t h e measurement o f l i n e w i d t h a t d i f f e r e n t t e m p e r a t u r e s i s n o t s u f f i c i e n t t o d i s t i n g u i s h t h e s e two l i m i t s . However, t h e c h e m i c a l s h i f t and t h e r e l a x a t i o n t i m e a t s i t e " B " , AWg and ( l - ) N ^ / f g , can be d e t e r m i n e d i n d e p e n d e n t l y f r o m c o n c e n t r a t i o n dependence o f t h e o b s e r v e d Net c h e m i c a l s h i f t and Net l i n e b r o a d e n i n g r e s p e c t i v e l y . ( 1 / T J M C T S e t uAv - f "L~' f o r b r e v i t y - , T B i f l i m i t ( c ) h o l d s , t h e n 1 K < 1 TrAv_ > ; > _ 1 _ T B < ^ AWg 2 > 9 > * A V i f l i m i t (d) h o l d s , t h e n — » j— TTAV _J T B '2B ... 2 ITAV AWg T h u s , i f t h e l i n e w i d t h as f g 1 i s l a r g e r t h a n t h e f r e q u e n c y s e p a r a t i o n o f t h e r e s o n a n c e f o r t h e two s i t e s , l i m i t ( c ) h o l d s ; i f t h e l i n e w i d t h as f g -»- 1 i s s m a l l e r t h a n AWg, l i m i t (d) h o l d s . T h i s e x t r a t r e a t m e n t o f t h e d a t a on t o p o f the t e m p e r a t u r e - 9 -control experiment will leave no ambiguity in distinguishing which process dominates the line broadening, and thus will ensure the correct interpretation of the data. With the above consideration in mind, one can systematically sortout the problem of chemical exchange at different limits, see Table 1.1 and Figure 1.1 Table 1.1 process domi nated cases AWg Av T by limits A, (a) ^ 0 + t TB TB y > T 2 B (b) ~o 4- T 2 B TB K < T 2 B B,(a) & (.b) '+ TB (c) to 4- 2 irAv/AWg« 1 ITAV V W B ' TB y > T 2 B (d) 4- t 2 TTAV/ AWg>> 1 TTAV T 2 B TB K < T 2 B Some extra information is provided by the last column of Table 1.1 in regard to an upper or lower limit of Tg . For example, we"can determine Tg through the measurement of line width at different concentrations, even i f we cannot determine T 2g as in case A(a). Tg itself is an upper limit for T 2 g. This could be important for the estimation of correlation time at site "B" or the distance between site "B" and another site which contributes most of the dipole-dipole relaxation between these two sites, as we will see later. -10-Computer s i m u l a t i o n o f the t e m p e r a t u r e b e h a v i o r o f T. 60 MHz, 100 MHz, and 220 MHz With AWB= 100 Hz a t 23.4 KGauss C h e m i c a l Exchange L i m i t Dominant P r o c e s s A l I 2 f 1 N2 , 1 N2 1 AWB o r {-j—) > (——) —-B 12B T B T B - n -| 1 1 1 1 I 1 1 I I Too 48 4X) 3.2 2A 1.6 0.8 1 0 0 0 / T - 12 -C. C h e m i c a l s h i f t s and c h e m i c a l exchange The o b s e r v e d c h e m i c a l s h i f t i n t h e p r e s e n c e o f c h e m i c a l 2 exchange was d e r i v e d by S w i f t and C o n n i c k under t h e c o n d i t i o n I f t h e exchange r a t e i s much l a r g e r t h a n t h e c h e m i c a l s h i f t d i f f e r e n c e a t s i t e "A" and "B", and s i t e "A" i s i n g r e a t e x c e s s o v e r s i t e "B", t h e o b s e r v e d c h e m i c a l s h i f t w i l l be T h i s c o r r e s p o n d s t o c a s e s B ( c ) and B(d) i n T a b l e l . T . C l e a r l y AWg c a n be e s t i m a t e d w i t h o u t any d i f f i c u l t y . D. The mechanisms o f t h e r e l a x a t i o n p r o c e s s A c o n c i s e and c o m p r e h e n s i v e d e s c r i p t i o n o f d i f f e r e n t r e l a x a t i o n mechanisms can be found i n C h a p t e r 11 and 13 o f the book by C a r r i n g t o n ^ . Fo r t h e systems I s t u d i e d i n t h e absence o f p a r a m a g n e t i c  s p e c i e s , t h e i n t r a m o l e c u l a r d i p o l e - d i p o l e i n t e r a c t i o n whose magnitude i s m o d u l a t e d by r o t a t i o n a l d i f f u s i o n w i l l dominate t h e r e l a x a t i o n p r o c e s s ^ . In t h i s c a s e , t h e r e l a x a t i o n r a t e s a r e g i v e n by 1 obsd A 6 o b s d = 6 A + f B A W B (1.15) (1.16) - 1 3 -MJ /' OC 0 C Y where ( l / T g ) ^ ! 0 / T | ) . a r e t h e t r a n s v e r s e and l o n g i t u d i n a l r e l a x a t i o n r a t e s r e s p e c t i v e l y . P r o t o n i i s r e l a x e d by p r o t o n j , r . . i s t h e i n t e r n u c l e a r d i s t a n c e , Y i s t h e m a g n e t o g y r i c r a t i o f o r p r o t o n s , ' f i i s t h e p l a n k c o n s t a n t , WQ i s t h e r e s o n a n c e f r e q u e n c y f o r t h e o b s e r v e d n u c l e i , x ( r o t a t i o n a l c o r r e l a t i o n t i m e ) i s a measure o f t h e c t i m e i t t a k e s f o r t h e a x i s o f t h e p r o t o n - c a r b o n c h e m i c a l bond t o r e o r i e n t t h e o r d e r o f a r a d i a n , so x can be a measure o f c t h e f l e x i b i l i t y a t t h e s m a l l m o l e c u l e - b i n d i n g s i t e on a m a c r o m o l e c u l e . F o r systems i n t h e p r e s e n c e o f p a r a m a g n e t i c s p e c i e s , t h e e l e c t r o n - p r o t o n d i p o l a r i n t e r a c t i o n s t h a t depend upon t h e e l e c t r o n - p r o t o n d i s t a n c e and t h e c o n t a c t h y p e r f i n e i n t e r a c t i o n t h a t depends on t h e e l e c t r o n s p i n d e n s i t y a t t h e n u c l e u s w i l l be t h e two dominant r e l a x a t i o n mechanisms. The r e l a x a t i o n r a t e s can t h e n be d e s c r i b e d by t h e Solomon-8 9 Bloembergen e q u a t i o n ' , w i t h t h e a p p r o x i m a t i o n W j « W s . In f a c t , W$ = 650 Wj . 1 1 S ( S + D Y j g V r 3 x _ 1 3 x . T 2 M i s ( s + 1 ) Y i g B IV + 3TC + 13TC 1 ^ — ? " L c 77^7 T T ^ j - 14 -2 2 Q2 _ , T - 7r % 1 5 L l + W X V 1 + WS T c J 4. 1 S ( S + IIAI r—^Vrl ( 1-19) 3 h 1 1 T "S 'e where T and T a r e t h e c o r r e l a t i o n t i m e s f o r d i p o l a r and c e , h y p e r f i n e i n t e r a c t i o n s r e s p e c t i v e l y ; S, t h e e l e c t r o n s p i n quantum number; Y j , t h e n u c l e a r m a g n e t o g y r i c r a t i o ; r , t h e i o n o r " s p i n - l a b e l " ( u n p a i r e d e l e c t r o n ) - p r o t o n i n t e r n u c l e a r d i s t a n c e ; g, t h e e l e c t r o n i c "g" f a c t o r ; B , t h e Bohr magneton; Wj and Ws, t h e Larmor a n g u l a r p r e c e s s i o n f r e q u e n c y f o r t h e n u c l e a r , and e l e c t r o n s p i n s , r e s p e c t i v e l y ; and A, t h e h y p e r f i n e c o u p l i n g c o n s t a n t . The f i r s t t e r m i n eq. (1.18) and (1.19) r e p r e s e n t s t h e d i p o l a r ( t h r o u g h s p a c e ) c o n t r i b u t i o n and s e c o n d t e r m t h e s c a l a r ( t h r o u g h - b o n d s ) t o t h e r e l a x a t i o n r a t e s . D e p e n d ing on t h e s i t u a t i o n s , some f u r t h e r s i m p l i f i c a t i o n s can be made. T h i s w i l l be d i s u c s s e d s e p a r a t e l y a s r e q u i r e d i n e a c h c h a p t e r . In t h e p r e s e n c e o f p a r a m a g n e t i c s p e c i e s , t h e e f f e c t i v e c o r r e l a t i o n t i m e T f o r t h e d i p o l a r i n t e r a c t i o n ^ i s c d e t e r m i n e d by t h e i n t e r p l a y o f r o t a t i o n a l c o r r e l a t i o n t i m e , T , e l e c t r o n i c s p i n - l a t t i c e r e l a x a t i o n t i m e , T s > and r e s i d e n c e t i m e a t s i t e "B", T G - 15 -W h i c h e v e r i s t h e s h o r t e s t w i l l dominate t h e d i p o l a r m o d u l a t i o n p r o c e s s , s i n c e e a c h p r o c e s s c a u s e s a change i n magnitude o f t h e d i p o l e - d i p o l e i n t e r a c t i o n , and t h u s c o n t r i b u t e s s i m i l a r l y t o r e l a x a t i o n . The e f f e c t i v e c o r r e l a t i o n t i m e T g f o r the h y p e r f i n e i n t e r -a c t i o n ^ ( t h e i n t e r a c t i o n t h a t i s t r a n s m i t t e d t h r o u g h c h e m i c a l bonds r a t h e r t h a n t h r o u g h s p a c e ) i s 1 - = i _ .+ i _ ' - (1.21) T e T s B w i t h t h e same meanings f o r ~ and |— as above. T s B A s y s t e m a t i c way o f d e t e r m i n i n g w h i c h c o r r e l a t i o n t i m e d o m i n a t e s t h e m o d u l a t i o n p r o c e s s e s f o r t h e r e l a x a t i o n has been r e v i e w e d by C o h n l ^ . E. Methods o f measurement. 1. Measurement O f s p i n - l a t t i c e r e l a x a t i o n t i m e , T-j 12 13 T-| can be measured by T-j , a d i a b a t i c r a p i d p a s s a g e , s a t u r a t i o n r e c o v e r y ^ , p r o g r e s s i v e s a t u r a t i o n ^ and s p i n - e c h o s ^ For s p i n - e c h o s , which were employed h e r e , a l l t e c h n i q u e s f o r m e a s u r i n g T.j a r e based on f i r s t a l t e r i n g t h e m a g n e t i z a t i o n w h i c h l i e s p a r a l l e l t o t h e f i e l d d i r e c t i o n (+Z). With e i t h e r one o r more r e c t a n g u l a r p u l s e s o f o s c i l l a t i n g m a g n e t i c f i e l d s o f s t r e n g t h , s u f f i c i e n t t o cause t h e m a g n e t i z a t i o n ( i n i t i a l l y a l o n g t h e z - a x i s ) t o p r o c e s s by 90° a b o u t t h e x ' - a x i s , t o end up a l o n g t h e y ' - a x i s o f a r e f e r e n c e frame w h i c h r o t a t e s a t t h e Larmor f r e q u e n c y a b o u t t h e z - a x i s ( s e e ^ f o r d i s c u s s i o n o f t h e r o t a t i n g frame r e p r e s e n t a t i o n ) . Or, w i t h a 180° p u l s e which a l i g n s the m a g n e t i z a t i o n a n t i p a r a l l e l t o t h e f i e l d (-Z), one t h e n o b s e r v e s t h e r e - e s t a b l i s h m e n t o f t h e e q u i l i b r i u m - 16 -m a g n e t i z a t i o n i n t h e f i e l d d i r e c t i o n . O n l y two methods w h i c h have been a c t u a l l y t r i e d o r used w i l l be c o n s i d e r e d h e r e . (a) IT - ( t - 2Tr)n o r TT-(t - j • IT • ^-)n« ( F i g u r e 1.2) M o n i t o r i n g can be a c c o m p l i s h e d by a t r a i n o f "2ir" p u l s e s t h a t r o t a t e s t h e m a g n e t i z a t i o n v e c t o r one c o m p l e t e r e v o l u t i o n i n t h e Y'Z' p l a n e o f t h e r o t a t i n g frame o f r e f e r e n c e . As t h e m a g n e t i z a t i o n r o t a t e s t h r o u g h t h e +Y' and -Y 1 d i r e c t i o n , a s i g n a l i s i n d u c e d i n t h e d e t e c t i o n c o i l and a t t h e end o f each 360° p u l s e , t h e m a g n e t i z a t i o n v e c t o r i s l e f t p a r a l l e l t o t h e Z a x i s where i t c o n t i n u e s i t s l o n g i t u d i n a l r e c o v e r y towards e q u i l i b r i u m . I f s u f f i c i e n t l y s t r o n g r a d i o f r e q u e n c y f i e l d H . i s a v a i l a b l e , t h e n a number o f m o n i t o r i n g p u l s e s can be a p p l i e d d u r i n g one r e l a x -a t i o n p r o c e s s , and t h e e n v e l o p e o f t h e i n d u c e d s i g n a l peaks can be r e d u c e d t o y i e l d f r o m a s e m i l o g p l o t o f log(<Mz>) vs t i m e . T h i s method i s q u i t e e c o n o m i c a l i n t i m e , but i t s e r r o r i s a l s o b i g , s i n c e t h e r e w i l l be a c o m m u l a t i v e e r r o r when e a c h m o n i t o r i n g p u l s e i s n o t e x a c t l y 3 6 0 ° . (b) (TT - t - f ) ( F i g u r e 1.3) In t h i s c a s e , m o n i t o r i n g i s a c c o m p l i s h e d by a p p l y i n g a Y p u l s e a t d i f f e r e n t t a f t e r t h e TT p u l s e . F o l l o w i n g each (TT - t - Tj-) p u l s e sequence, a w a i t i n g p e r i o d l o n g e r t h a n 5T-j i s r e q u i r e d t o a l l o w t h e m a g n e t i z a t i o n , <Mz>,to r e t u r n t o i t s e q u i l i b r i u m v a l u e between e x p e r i m e n t s . A l t h o u g h t h i s method F i g u r e 1.2 Measurement o f L p Method #1 E f f e c t o f a 180° - ( t - 360°)n sequence o f p u l s e s o f o s c i l l a t i n g m a g n e t i c f i e l d (H^) on t h e m a g n e t i z a t i o n v e c t o r , M Q, i n a r o t a t i n g frame o f r e f e r e n c e X',Y',Z' As t h e m a g n e t i z a t i o n p a s s e s t h r o u g h t h e o b s e r v a t i o n a x i s ( y 1 ) , a s i g n a l i s i n d u c e d i n t h e r e c e i v e r c o i l , as shown i n t h e o s c i l l o s c o p e t r a c e s below. A. E q u i l i b r i u m B. I n i t i a l 180° H-j p u l s e a l o n g t h e x ' a x i s C. M a g n e t i z a t i o n r e t u r n toward + z' d i r e c t i o n D. & F. A 360° ( m o n i t o r i n g ) p u l s e a l o n g t h e x' a x i s t o make t h e m a g n e t i z a t i o n pass t h r o u g h t h e a x i s o f t h e d e t e c t o r ( y ' ) . E. F u r t h e r m a g n e t i z a t i o n r e c o v e r y t o + z; G. Complete r e c o v e r y o f z - m a g n e t i z a t i o n H. Diagram suhowing t h e e x p o n e n t i a l r e c o v e r y o f an i n i t i a l l y i n v e r t e d m a g n e t i z a t i o n v e c t o r , M Q. I. O s c i l l o - s c o p e p h o t o g r a p h o f p r o t o n n u c l e a r i n d u c t i o n f o r H 20 u s i n g a d i o d e ( i . e . , r e c t i f y i n g ) d e t e c t o r . Time base i s 0.5 sec/cm. Time between 360°.pulses a r e 200 m.s. M(t) = M 0 ( l -2e t / Ti) TT r h T" " i f k—•—•—•—1—&—1—i—i—i—i——i—i—i i i i i i_ 50 I-100 150 SECONDS - 19 -Figure 1.3 Measurement of T 1 : Method #2 Effect of 180° - t - 90° -pulse magnetic f ield sequence on the magnetization vector, MQ, in a rotating frame of reference X', Y', Z' and the corresponding induced magnetization behaviour appearing on an oscilloscope. _. A. Equilibrium B. Initial 180° h^-pulse along the X' axis C. Recovery of Mz towards +Z' directions D. 90° (monitoring) pulse along X' axis to rotate the magneti-zation to the axis (y1) of the detector E. Magnetization de-focusing in the X', Y' plane, (free-induction decay) due to inhomogeneity in HQ applied field F. Diagram showing the signal induced in the detector in the X'-Y' plane by a 90° pulse along the X'-axis varying delay times, t , t ' , t", etc., after an init ial 180° H-j-pulse. G. Oscilloscope photograph of proton nuclear induction from water in the presence of Mn++(10~3M/Jl) and trypsin (10"3M/£). The oscilloscope display was triggered immediately after the completion of a 90° monitoring pulse. Time base 1 m.s./cm. - 21 -has t h e drawback o f " w a i t i n g " , t h e d a t a a r e m u c h more r e l i a b l e . 2. Measurement o f s p i n - s p i n r e l a x a t i o n t i m e , TQ The s p i n - s p i n r e l a x a t i o n t i m e s o f t h e s m a l l m o l e c u l e s i n s o l u t i o n a r e a l w a y s overshadowed by t h e c o n t r i b u t i o n s f r o m s o u r c e s l i k e f i e l d i n h o m o g e n e i t y , e f f e c t o f d i f f u s i o n , e t c . T h e s e u n f a v o r a b l e c o n t r i b u t i o n s can be removed by r e f o c u s i n g t h e phases o f t h e s p i n s i n t h e x'-y' p l a n e w i t h t h e t e c h n i q u e o f s p i n e c h o . T h i s i s n e c e s s a r y , b e c a u s e t h e i n t r a m o l e c u l a r c o n t r i b u t i o n T 1 _ T 2 o f methyl group might be o n l y one q u a r t e r o f t h e c o n t r i -b u t i o n f r o m f i e l d i n h o m o g e n e i t y . T h e r e a r e a t l e a s t h a l f a dozen ways t o d e t e r m i n e T^ t h r o u g h 18 t h e s p i n echo e x p e r i m e n t . G i l l - M e i b o o m m o d i f i c a t i o n o f C a r r -P u r c e l l sequence i s t h e t e c h n i q u e most o f t e n used f o r t h e s t u d i e s i n l i q u i d s . 19 (a) C a r r - P u r c e l l sequence - w i t h G i l l - M e i b o o m m o d i f i c a t i o n ( F i g u r e 1.4) T h i s sequence c o n s i s t s o f a |- p u l s e a l o n g t h e X ' - a x i s f o l l o w e d by a s e r i e s o f (90° phase s h i f t e d ) TT p u l s e s a l o n g t h e Y 1 a x i s o f t h e r o t a t i n g r e f e r e n c e f r a m e . In t h i s way one can m i n i m i z e t h e c o n t r i b u t i o n o f f i e l d i n h o m o g e n e i t y which c a u s e s t o be t h e T 2 A m u c h s h o r t e r , and i t can a l s o m i n i m i z e t h e e r r o r on s e t t i n g t h e p u l s e d u r a t i o n s . The v a l u e o f T 2 can e a s i l y be c a l c u l a t e d from t h e e n v e l o p e o f t h e e c h o s d e v e l o p e d a t F i g u r e 1.4 Measurement o f N u c l e a r T, E f f e c t o f a m o d i f i e d C a r r - P u r c e l l p u l s e sequence on t h e m a g n e t i z a t i o n v e c t o r , M Q . A. e q u i l i b r i u m B. 90° p u l s e a l o n g t h e x ' - a x i s C. F r e e - i n d u c t i o n d e c a y D. 90°-phase-shifted 180° p u l s e a l o n g t h e y ' - a x i s c a u s i n g t h e r e f o c u s i n g o f t h e f i e l d - i n h o m o g e n u i t y - i n d u c e d " f a n n i n g - o u t " o f i n d i v i d u a l m a g n e t i z a t i o n v e c t o r s E. Echo ( c u l m i n a t i o n o f r e - f o c u s i n g ) F. F r e e - i n d u c t i o n d e c a y G. 180° - p u l s e a l o n g y ' - a x i s H. echo I. d i a g r a m showing t h e e x p o n e n t i a l d e c a y o f t h e a m p l i t u d e o f t h e echoes i n t h e x'y' p l a n e : t h i s d e c a y e n v e l o p e r e p r e s e n t s t h e " t r u e " i n h e r e n t 1^ f o r t h e p r o t o n , c o r r e c t e d f o r i n h o m o g e n u i t y i n H . o J . O s c i l l o s c o p e p h o t o g r a p h o f t h e p r o t o n n u c l e a r m a g n e t i z a t i o n f o r w a t e r i n t h e p r e s e n c e o f M n + + ( 1 0 ~ 4 M / J I ) and t r y p s i n (10~ 3M/&). Time base i s 10 ms/cm. Time between s u c c e s s i v e 180° p u l s e s 2 ms. The lo w e r t r a c e i s t h e f r e e - i n d u c t i o n d e c a y w i t h no a t t e m p t t o e l i m i n a t e t h e c o n t r i b u t i o n f r o m f i e l d i n h o m o g e n e i t y ( i . e . , no 180° p u l s e s ) . K. O s c i l l o s c o p e t r a c e o f p r o t o n n u c l e a r m a g n e t i z a t i o n o f w a t e r , u s i n g a l o n g e r t i m e between 180° p u l s e s o f 0.2 s e c . Time base i s 0.5 sec/cm. - 23 -- 24 -d i f f e r e n t t i m e s . (b) l i n e w i d t h measurement f r o m a c o n t i n u o u s - w a v e a b s o r p t i o n s i g n a l I f t h e l i n e w i d t h o f t h e sample i s much l a r g e r t h a n t h e f i e l d i n h o m o g e n e i t y , t h e n 1^ can be e s t i m a t e d as 1/irAv. AV i s t h e f u l l l i n e w i d t h o f t h e r e s o n a n c e l i n e i n Hz a t h a l f h e i g h t o f t h e r e s o n a n c e l i n e . Sometimes we w i l l be i n t e r e s t e d i n t h e changes o f t h e ' l i n e w i d t h i n t r o d u c e d by d i f f e r e n t p e r t u r b a t i o n s , i n the absence and p r e s e n c e o f enzymes, f r e e r a d i c a l s , m e t a l s , e t c . In t h i s c a s e , we can d e t e r m i n e t h e changes i n l i n e w i d t h by e m p l o y i n g an i n t e r n a l s t a n d a r d which w i l l n o t be e f f e c t e d by t h e p e r t u r -b a t i o n s ; t h e n o n - s p e c i f i c l i n e - b r o a d e n i n g can t h u s be removed by s u b t r a c t i n g t h e l i n e w i d t h o f i n t e r n a l s t a n d a r d l i n e from t h a t o f t h e l i n e o f i n t e r e s t . 20 3. F o u r i e r - T r a n s f o r m NMR ( F i g u r e 1.5) A s u f f i c i e n t l y s t r o n g r . f . p u l s e w i l l c a u s e t h e m a g n e t i z a -t i o n v e c t o r s o f a l l t h e n u c l e i o v e r a s p e c t r a l f r e q u e n c y range o f r o u g h l y YH^/2TT t o t i p by ( f o r example) 9 0 ° , and t h e n p r e c e s s i n t h e X'Y' p l a n e , g e n e r a t i n g a complex b e a t f r e q u e n c y p a t t e r n i n t h e d e t e c t o r c o i l , w h i c h i s l o c a t e d i n t h e x'-y' p l a n e . T h i s p a t t e r n c o n s i s t s o f a s u p e r p o s i t i o n o f a l l t h e f r e e i n d u c t i o n d e c a y s ( F I D s ) o f each o f t h e l i n e s i n t h e s p e c t r u m . - 25 -F i g u r e 1.5 T r a n s i e n t and F o u r i e r - T r a n s f o r m e d NMR Responses F o r an I n h i b i t o r o f T r y p s i n A. The d e c a y i n g p a t t e r n o f i n t e r f e r r i n g f r e q u e n c i e s i n I t i s t h i s p a t t e r n w hich s t o r e d i n t h e d i g i t a l computer. B. The f o u r i e r t r a n s f o r m o f t h e t r a n s i e n t r e s p o n s e y i e l d s a s p e c t r u m w h i c h i s e q u i v a l e n t t o an o r d i n a r y slow-sweep c o n t i n u o u s wave a b s o r p t i o n s p e c t r u m , sample: p-methoxyl p h e n y l g u a n i d i n e . H C l - . t h e t i m e domain, f o l l o w i n g a p p l i c a t i o n o f a 90° H, - p u l s e . (0.C5 M / l ) . A c q u s i t i o n t i m e 4 s e c . Sweep w i d t h 1000 Hz. Number o f t r a n s i e n t s 1. I I I I I I I I I I I I I I I I I I I I I[I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I II 1 I I I ,1 I I I I I , I - 27 -Each FID w i l l d ecay a c c o r d i n g t o i t s own r e l a x a t i o n t i m e and w i l l have an a m p l i t u d e p r o p o r t i o n a l t o t h e p o p u l a t i o n o f t h a t p a r t i c u l a r n u c l e u s i n t h e x ' y ' - p l a n e . T h i s d e c a y i n g p a t t e r n o f i n t e r f e r r i n g f r e q u e n c i e s i s t h e n s t o r e d i n a m u l t i - c h a n n e l d i g i t a l computer u s i n g a n a l o g t o d i g i t a l c o n v e r s i o n i n t h e t i m e domain. The F o u r i e r t r a n s f o r m o f t h i s i n f o r m a t i o n f r o m t h e time domain i n t o the f r e q u e n c y domain w i l l r e s u l t t h e e q u i v a l e n t o f an o r d i n a r y slow-sweep c o n t i n u o u s - w a v e a b s o r p t i o n s p e c t r u m . The a d v a n t a g e s o f t h i s t e c h n i q u e o v e r c o n t i n u o u s wave mode a r e a. i n f o r m a t i o n can be accummulated a t a much f a s t e r r a t e ; so t h a t c h e m i c a l l y d i l u t e samples can be s t u d i e d u s i n g t i m e - a v e r a g i n g . b. u n d i s t o r t e d l i n e shapes and t r u e s p e c t r a l f r e q u e n c i e s a r e o b t a i n e d . c. T-| o f a l l t h e l i n e s i n the s p e c t r u m can be measured a t t h e same t i m e by c a r r - p u r c e l l s e q u e n c e . d. n o n - s p e c i f i c b r o a d e n i n g can be removed a l m o s t c o m p l e t e l y , by i n c l u s i o n o f an i n e r t s p e c i e s i n the same sample t u b e . In s u c c e e d i n g c h a p t e r s , t h e methods i n t r o d u c e d i n t h i s c h a p t e r w i l l be a p p l i e d t o a number o f a s p e c t s o f b i n d i n g o f M n + + o r s p e c i f i c i n h i b i t o r s t o the enzyme, t r y p s i n , i n c l u d i n g : number o f b i n d i n g s i t e s , k i n e t i c s and s t r e n g t h o f b i n d i n g , - 28 -r i g i d i t y a t t h e b i n d i n g s i t e , and d i s p o s i t i o n o f t h e b i n d i n g s i t e r e l a t i v e t o o t h e r s i t e s . R e f e r e n c e s 1. H.M. M c C o n n e l l , J . Chem. Phys. 28, 430 ( 1 9 5 8 ) . 2. T . J . S w i f t and R.E. C o n n i c k , J . Chem. Phys. 37, 307 ( 1 9 6 2 ) . 3. Z. Luz and S. Meiboom, J . Chem. Phys. 40, 2686 ( 1 9 6 4 ) . 4. B.D. S y k e s , P.G. S c h m i d t , G.R. S t a r k , J . Amer. Chem. Soc. 245, 1180 ( 1 9 7 0 ) . 5. J.A. P o p l e , W.G. S c h n e i d e r , and H.J. B e r n s t e i n , H i g h r e s o l u t i o n n u c l e a r m a g n e t i c r e s o n a n c e , McGraw H i l l book company, N.Y., p.204 ( 1 9 5 9 ) . 6. A C a r r i n g t o n and A.D. M c L a c h l a n , H a r p e r & Row P u b l i s h e r , N . Y . , 0 9 6 7 ) . 7. P.G. S c h m i d t , G.R. S t a r k and J.D. B a l d e s c h w i e l e r , J . o f B i o l . Chem., 244, 1860 ( 1 9 6 9 ) . 8. I . Solomon, Phys. Rev., 99_, 559, ( 1 9 5 5 ) . 9. N. Bloembergen, J . Chem. Phys. 27, 572, ( 1 9 5 7 ) . 10. A.S. M i l d v a n and M. Cohn.Advance. Enzymol. 33. 1 ( 1 9 7 0 ) . 11. C P . S l i c h t e r , P r i n c i p l e s o f M a g n e t i c R e s o n a n c e , C h a p t e r 2, H a r p e r & Row, N.Y. ( 1 9 6 3 ) . 12. B.D. S y k e s , J.A.C.S. 9J_, 949 ( 1 9 6 9 ) . 13. L.E. D r a i n , P r o c . Phys. S o c . (London) 62A, 301 ( 1 9 4 9 ) . - 29 -14. A.L. Van Geet and D.N. Hume, A n a l y t . Chem. 37., 983 ( 1 9 6 5 ) . 15. A.L. Van Geet and D.N. Hume, A n a l y t . Chem. 37_, 979 ( 1 9 6 5 ) . 16. E.L. Hahn, Phys. Rev. 80, 580 ( 1 9 5 0 ) . 17. H . Y . C a r r and E.M. P u r e e ! ! , Phys. Rev. 94, 630 (1 9 5 4 ) . 18. B r u k e r ' s Lab. Manu., "NMR p u l s e d s p e c t r o m e t e r s " , 19. S. Meiboom and D. G i l l , Rev. S c i . I n s . 29_, 688 (1 9 5 8 ) . 20. T.C. F a r r a r and E.D. B e c k e r , " P u l s e d and F o u r i e r T r a n s f o r m NMR." Academic P r e s s , ( 1 9 7 1 ) . - 30 -CHAPTER I I M a g n e t i c Resonance S t u d i e s o f M n ( II) i o n s B i n d i n g t o T r y p s i n A. I n t r o d u c t i o n The p u r p o s e o f t h e work h e r e was t w o f o l d : f i r s t , t o d e t e r m i n e t h e s t o i c h i o m e t r y , b i n d i n g c o n s t a n t s , and r i g i d i t y o f bound'water, i n t h e b i n d i n g o f M n + + t o a c t i v e t r y p s i n , as an a n a l o g y w i t h t h e i n t e r a c t i o n o f C a + + w i t h t r y p s i n and s e c o n d , t o f i n d a method f o r m e a s u r i n g t h e r o t a t i o n a l l a b i l i t y o f w a ter bound n e a r a p a r a m a g n e t i c s i t e on a m a c r o m o l e c u l e which i s more * 1 2 d i r e c t t h a n use o f "enhancement f a c t o r s " * . C a l c i u m ( I I ) i o n s have a number o f e f f e c t s on t h e p r o p e r t i e s ++ 3 o f t r y p s i n : Ca s t a b i l i z e s t r y p s i n a g a i n s t a u t o l y s i s , 4 i n c r e a s e s enzyme s t a b i l i t y toward a c i d , b a s e , o r u r e a , promotes 5 t h e f o r m a t i o n o f a c t i v e t r y p s i n f r o m t r y p s i n o g e n , and enhances * 2 NOTE: E i s i n g e r e t a l . . The o b s e r v e d enhancement i n t h e r e l a x a t i o n r a t e s l/li and 1/T 2 i n terms o f p a r a m e t e r s zi and e 2 , i s d e f i n e d as f o l l o w s : C o n t r i b u t i o n o f M n (II) i o n s , i n t h e p r e s e n c e o f macro-^ . m o l e c u l e s t o t h e s p i n r e l a x a t i o n r a t e o f w a t e r p r o t o n s C o n t r i b u t i o n o f an e q u a l c o n c e n t r a t i o n o f M n (II) i o n s , i n t h e a b s e n c e o f m a c r o m o l e c u l e s , t o t h e s p i n r e l a x a t i o n r a t e o f t h e w a t e r p r o t o n s . - 31 -the efficiency of catalysis toward benzoyl-L-arginine ethyl ester (BAEE) . These effects may also be produced by the div-alent metals, Mn, Cd, Co, and by trivalent Nd .^ Among these, Mn possesses the dual advantages that i t has an observable electron spin resonance (ESR) spectrum and, in addition, has a well-described effect on the nuclear magnetic relaxation of protons in-water molecules. In this paper, we use the EPR peak p height combined with a Scatchard analysis to determine the number of strong and weak Mn -binding sites on trypsin and their respective binding constants; the nuclear magnetic resonance (NMR) data may then be analyzed to yield the rotational corre-lation time for bound water, a measure of the degree of immobili-zation of water at the Mn++-binding site. Since the effect of Mn++ in enhancing catalysis of BAEE by trypsin is virtually identical to that of Ca + + , the present results should be directly applicable toward understanding of the effect of Ca + + on trypsin. B. Theory The most convincing derivation of the strength and number 1 ++ of binding sites for Mn on a macromolecule using electron 8 9 spin resonance data is by means of a Scatchard plot ' . This procedure has been used independently by Birkett et a l . as described in a recent review by Dwek,^ and is described in Results and Discussion, part 1. - 32 -R e d u c t i o n o f t h e NMR d a t a i s f a r l e s s s t r a i g h t f o r w a r d . Water m o l e c u l e s i n t h e p r e s e n t s t u d i e s may r e s i d e a t any o f t h r e e d i s t i n c t s i t e s : f r e e w a t e r ( A ) , h y d r a t e d f r e e M n + + i o n ( B ) , and w a t e r bound t o t h e Mn:enzyme complex ( C ) , and a g i v e n w a t e r m o l e c u l e may jump from any s i t e t o any o t h e r s i t e , a l t h o u g h a l l p r e v i o u s a n a l y s e s 1 0 have been based on a s i m p l i f i e d mechanism i n which one o f t h e exchange p r o c e s s e s i s i g n o r e d : 1 1 S w i f t and C o n n i c k ' s c a l c u l a t i o n s o f 1^ f o r t h e system , (2.1) 12 have r e c e n t l y been g e n e r a l i z e d by Degani and F i a t t o a t r u e t h r e e - s i t e s i t u a t i o n and s i n c e t h e o r e t i c a l i n t e r p r e t a t i o n o f i s much s i m p l e r t h a n t h a t o f when p a r a m a g n e t i c s p e c i e s a r e 13 p r e s e n t , we now p r e s e n t t h e d e r i v a t i o n o f f o r t h e t h r e e - s i t e s y s t e m , ( 2 . 2 ) . F o r t h e s y s t e m , (2.2) and ( 2 . 3 ) , M c C o n n e l l ' s 14 e q u a t i o n s may be w r i t t e n , (2.1) (2.2) A + M v B where M i s metal i o n and B + E v, ^ C K-2 E i s enzyme, and ME (2.3) A + ME C -3 i s metal-enzyme complex, - 33 -dM. A d t fll/.-V ( k ^ M ] + k 3 [ M E ] ) M z A + k _ 1 M z B + k _ 3 M z C dM. d t + K ^ M l M ^ - (k_, + k 2 [ E ] ) M zB + k _ 2 M z C dM 1 ~ d T c M c •z - M o + k 3 [ M E ] M z A + k 2 [ E ] M z B - ( k _ 2 + k _ 3 ) M z C (2.4) By s o l v i n g t h e syste m , (2.4), s u b j e c t t o t h e i n i t i a l c o n d i t i o n o f a 180° p u l s e , t h e f o l l o w i n g r e l a t i o n may be d e r i v e d , a more d e t a i l e d d e r i v a t i o n i s p r e s e n t e d i n Appendix A. 1 1_ _ 1 T l " T7 1-r c + k - 2 + k - 3 ' l ( J _ + k ^ + k 2 [ E ] ) ( - l - + k _ 2 ± k _ 3 ) - k _ 2 k 2 [ E ] T i T r k - l + F T ^TT + k - l + k - 2 ^ E ^ ~L ' l / - 3 k ^ M ] 1- ^-2 T { T + k - l + k 2 [ E ] ) ( - l T + k-2 + k - 3 } ~ k _ 2 k 2 [ E ]  T l T l k - l + F T ( ~ B - + k - l + k 2 [ E ] ) ~L ' l k 3 [ M E ] , (2.5) - 34 -There are two conditions under which the general expression, ( 2 . 5 ) , is rendered tractable. First, i f the Av±B and A ^ C processes are both fast compared to the largest of ( l/T^), B C (1/T-| ), and (l/T-j ), then the observed longitudinal relaxation is independent of the Bv^C process, and ( 1 / V o b s = ( 1 / T 1 A ) + f B ( 1 / T l B ) + F C ( 1 / T l C ) ( 2 ' 6 ) where f. is the fraction of water molecules at each site and f^  = 1 for free water because i t is present in such large excess. On the other hand, i f the B^C process is slow compared to A?=^ B and AF^C, then ^ V o b s = ( 1 / T 1 A > + V < T 1 B + TB> + V ( T 1 C + TC> (2.7) where T. is the lifetime for water at the i 'th site. It is in fact likely that both simplifying conditions apply in the present experiments: the exchange rate for water solvated to Mn++ is fast and shows only slight dependence on + + 1 5 the degree of substitution about the Mn atom; moreover, the rate of dissociation of Mn++ from the Mn:enzyme complex is ++ probably slower than the dissociation of water from an Mn ion. These views will be supported in the discussion. - 35 -C. E x p e r i m e n t a l A n a l y t i c a l g rade M n C ^ ^ h ^ O was o b t a i n e d f r o m B r i t i s h Drug Houses. T w i c e l y o p h i l i z e d and s a l t - f r e e t r y p s i n was p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n and used w i t h o u t f u r t h e r p u r i f i c a t i o n . T r y p s i n a c t i v i t y was d e t e r m i n e d by t i t r a t i o n w i t h p - n i t r o p h e n y l , p * - q u a n i d i n o b e n z o a t e ; ^ t h e enzyme was fou n d t o be 50% a c t i v e . M n + + s o l u t i o n s were made up t o c o n c e n t r a t i o n s o f [ M n + + ] = 1 0 ' 1 , 1 0 " 2 , 5 x l 0 " 3 , 1 0 " 3 , 5 x l 0 ~ 4 , 2 . 5 x l 0 " 4 , and 10" 4M, w i t h each s o l u t i o n s 0.05M i n t r i s - m a l e a t e b u f f e r , pH 7.1. F o r measurements i n p r e s e n c e o f enzyme, 12 mg o f t r y p s i n was d i s s o l v e d i n 0.5ml o f each o f t h e M n + + s o l u t i o n s . ESR measurements. A V a r i a n model E-3 s p e c t r o m e t e r o p e r a t i n g a t 9.5 GHz p r o v i d e d a l l ESR d a t a ( F i g u r e 2 . 1 ) . F r e e [ M n + + ] c o n c e n t r a t i o n was d e t e r m i n e d from t h e a m p l i t u d e o f a p a r t i c u l a r Mn ESR t r a n s i t i o n , c a l i b r a t e d from samples o f known M n + + c o n c e n -t r a t i o n . [The a m p l i t u d e o f t h e ESR s i g n a l f o r Mn bound t o 19 enzyme w i l l be n e g l i g i b l e . ] To e n s u r e u n i f o r m i t y o f sample s i z e , a group o f t u b e s o f e q u a l d i a m e t e r were s e l e c t e d f r o m a b a t c h o f a p p r o x i m a t e l y 1.5 mm m e l t i n g - p o i n t c a p i l l a r y t u b e s . Each ESR peak h e i g h t r e p r e s e n t s t h e a v e r a g e o f a t l e a s t 6 s e p a r a t e d e t e r m i n a t i o n s . - 36 -F i g u r e 2.1 E.S.R. sp e c t r u m o f 1 0 " 4 M/JJ, M n + + s o l u t i o n a t pH 7.1 and 2 1 ± 0 . 5 ° C . The s p e c t r u m was r e c o r d e d a t 10 mW (microwave) power and 2.5 Gauss m o d u l a t i o n a m p l i t u d e . The r e s o n a n c e l i n e peak h e i g h t used f o r e s t i m a t i o n o f f r e e M n + + c o n c e n t r a t i o n was t h e t h i r d l i n e f r o m t h e r i g h t . M a g n e t i c F i e l d 3400 G. Microwave power lOmW M o d u l a t i o n a m p l i t u d e 2.5 G. 5 R e c e i v e r G a i n 1.25 x 10 R e c o r d e r t i m e . c o n s t a n t 1 s e c . Scan t i m e 8 min 3 Scan r a n g e 10 Gauss - 38 -NMR measurements. (a) B r u k e r p u l s e s p e c t r o m e t e r . T h i s s p e c t r o m e t e r c o n t a i n s a b a s i c 1 MHz q u a r t z O s c i l l a t o r w i t h a f r e q u e n c y s t a b i l i t y l y i n g between 0.01-0.001 Hz. T h r e e s e p a r a t e p u l s e c h a n n e l s a r e a v a i l a b l e t o g a t e t h e h i g h f r e q u e n c y i n t h e o s c i l l a t o r u n i t . The b a s i c 1 MHz f r e q u e n c y f r o m t h e main o s c i l l a t o r i s t a k e n i n t o a f r e q u e n c y s y n t h e s i z e r , where h a l f o f t h e r e s o n a n c e f r e q u e n c y i s p r o d u c e d . T h i s h a l f h i g h f r e q u e n c y i s t h e n f e d t o t h e t h r e e c h a n n e l s a f t e r a m p l i f i c a t i o n . The f i r s t c h a n n e l i s g a t e c h a n n e l I where t h e h i g h f r e q u e n c y s i g n a l i s f e d a f t e r b e i n g phase s h i f t e d , d o u b l e d and a m p l i f i e d . In t h e second c h a n n e l , w hich i s g a t e c h a n n e l I I , t h e h i g h f r e q u e n c y i s f e d d i r e c t l y a f t e r b e i n g d o u b l e d and a m p l i f i e d . In t h e t h i r d c h a n n e l , t h e r e f e r e n c e c h a n n e l , t h e h i g h f r e q u e n c y f e d s e r v e s as a phase c o h e r e n t r e f e r e n c e f r e q u e n c y f o r t h e phase s e n s i t i v e d e t e c t o r . The g a t e c h a n n e l I i s opened o n l y by p u l s e I and t h e g a t e c h a n n e l I I i s opened by p u l s e s I I and I I I . The h i g h f r e q u e n c y i n t h e f o r m o f p u l s e s a f t e r p a s s i n g t h r o u g h t h e g a t e s opened up by d.c. p u l s e s I , I I , and I I I i s l e d t h r o u g h a f i n e s t a g e a m p l i f i e r t o t h e t r a n s m i t t e r c o i l ( s i n g l e c o i l ) i n t o t h e p r o b e where i t e x c i t e s t h e NMR f r e q u e n c y s i g n a l . This NMR f r e q u e n c y s i g n a l combined w i t h t h e s i g n a l p r o d u c e d by r . f . p u l s e s i s p a s s e d t h r o u g h a p r e a m p l i f i e r and a f t e r a t t e n u a t i o n i s d e t e c t e d by t h e r e c e i v e r e i t h e r by d i o d e o r phase s e n s i t i v e d e t e c t i o n . The maximum band w i d t h o f t h e r e c e i v e r i n the s p e c t r o m e t e r - 39 -is 1 MHz and i t can be reduced to 100 KHz. The dead time of the receiver after an r.f. pulse is approximately 5-6 usee. The experiments in this study were done using a band width of 1 MHz. The magnet used for polarizing magnetic f ield Ho was Varian DP-60, 12 inch diameter pole gap, high-resolution electro-magnet. The signal amplitudes were recorded on a Tektronix Type 549 storage oscilloscope (band width 30 MHz) with type 1A1 Dual Trace plug in unit. (b) Measurement of relaxation time. T-j was measured by a 180° — T — 90° pulse sequence and T 2 by the Gill-Meiboom modifi-18 cation of a Carr-Purcell sequency . A short review on these techniques was provided in chapter 1. Each reported T-j or T 2 represents an average of at least 3 independent determinations, Temperature control to 21 ± 0.5°C (room temperature) was achieved with a Bruker temperature contol unit, B-ST 100/700. Flat-ended 8mm sample tubes were used to reduce inhomogeneity. D. Results and Discussion 1. Strength and number of Mn++-binding sites on trypsin. Since the electron paramagnetic resonance (EPR) signal for free Mn++ is narrow and easily observed, while the EPR signal for Mn bound to a macromolecule is broadened beyond detection, the EPR peak height for Mn in the presence of trypsin provides - 40 -a measure o f the. c o n c e n t r a t i o n o f f r e e manganous i o n , [Mn]^. The t o t a l [ M n ] Q c o n c e n t r a t i o n and t o t a l enzyme [ E ] a r e known, so t h a t t h e EPR d a t a can be combined t o g i v e t h e c o n c e n t r a t i o n o f "bound" manganese, [Mn]^. With t h e h e l p o f 3 d i m e n s i o n a l s t r u c t u r e 20 o f D I P - t r y p s i n a d i r e c t and r i g o r o u s d e t e r m i n a t i o n o f the number and s t r e n g t h o f b i n d i n g s i t e s f o r metal t o enzyme can be f u r n i s h e d 8 9 by a S c a t c h a r d ' r e d u c t i o n o f EPR peak h e i g h t d a t a f o r a s e t o f s o l u t i o n s o f v a r y i n g [ M n ] Q i n t h e p r e s e n c e o f a c o n s t a n t enzyme l e v e l , [ E ] Q , as f o l l o w s . The n o n - l i n e a r b e h a v i o u r o f t h e d a t a i n a ' S c a t c h a r d p l o t ( F i g u r e 2.2) c l e a r l y shows t h a t t h e r e e x i s t s a t l e a s t two k i n d s o f b i n d i n g s i t e s on t r y p s i n and t h e s t r o n g e r b i n d i n g s i t e has an i n t e r c e p t on t h e x - a x i s o f a b o u t 0.5. S i n c e "I c NPGB a c t i v e s i t e t i t r a t i o n showed t h a t 50% o f t h e t r y p s i n was f u l l y a c t i v e i t can be c o n c l u d e d t h a t o n l y t h e a c t i v e t r y p s i n can have a s t r o n g b i n d i n g s i t e . F o r s i m p l i c i t y , we assume t h a t t h e r e a r e j u s t two t y p e s o f M n + + - b i n d i n g s i t e s , w i t h r e s p e c t i v e maximum o c c u p a n c y n-j and n 2 and r e s p e c t i v e b i n d i n g c o n s t a n t K-| and K 2. With t h i s a s s u m p t i o n , t h e EPR peak h e i g h t d a t a p r o v i d e c o n c e n t r a t i o n v a l u e s f o r f i t t i n g t o t h e e q u a t i o n , V = Mn, - v) + Mn 9 " v) (2.8) [ M n ] f where v = (2.9) - 41 -F i g u r e C a p t i o n s F i g u r e 2.2 S c a t c h a r d p l o t f o r d e t e r m i n a t i o n o f s t r e n g t h and number o f b i n d i n g s i t e s f o r M n + + t o t r y p s i n . S o l i d l i n e i s a n o n - l i n e a r l e a s t - s q u a r e s f i t t o c o n c e n t r a t i o n d a t a d e r i v e d f r o m EPR peak h e i g h t measurements ( s e e t e x t ) ; t h e two d o t t e d l i n e s r e p r e s e n t a decom-p o s i t i o n o f t h e b i n d i n g i n t o two t y p e s o f s i t e s w i t h ( maximum o c c u p a n c y g i v e n by t h e x - i n t e r c e p t f o r each l i n e and ( n e g a t i v e ) b i n d i n g c o n s t a n t g i v e n by t h e s l o p e o f each l i n e . - 43 -20 w i t h n-j = 0.5, 3 < n 2 < 4 . 5 ( f r o m 3 d i m e n s i o n D I P - t r y p s i n , see b e l c v and [Mn]^. a r e v a l u e s d e t e r m i n e d by e x p e r i m e n t s . The computer i s r e a d i l y programmed t o p e r f o r m n o n - l i n e a r l e a s t - s q u a r e s f i t * o f e q. (2.8) t o the c o n c e n t r a t i o n d a t a d e r i v e d and f r o m EPR peak h e i g h t s , A g a v e t h e r e s u l t shown as t h e s o l i d c u r v e i n F i g u r e 2.2. The x - i n t e r c e p t f o r each d o t t e d l i n e g i v e s t h e maximum number o f M n + + a t t h a t s i t e , w h i l e t h e y - i n t e r c e p t f o r e a c h l i n e g i v e s t h e v a l u e o f nK f o r t h a t s i t e . The two s t r a i g h t ( d o t t e d ) l i n e s i n F i g u r e 2.2 thus r e p r e s e n t t h e d e c o m p o s i t i o n o f t h e r e s u l t s i n t o t h e two s e p a r a t e terms on t h e r i g h t - h a n d - s i d e o f eq. ( 2 . 8 ) . The b e s t f i t was f o u n d f o r n 2 = 4.5, K-, = 2900 l i t e r m o l e " 1 , and K 2 = 53 l i t e r m o l e " 1 . I n s p e c t i o n o f t h e x - r a y s t r u c t u r e o f Of) D I P - t r y p s i n shows t h a t the most l i k e l y l o c a t i o n f o r t h e s t r o n g b i n d i n g s i t e would be between Asp 153 and Asp 71. For t h e n a t i v e t r y p s i n , weak b i n d i n g s i t e s a r e l i k e l y a t n e a r b y G l u 77 o r t h e remote G l u 186. F o r t h e i n a c t i v e enzyme, t h e s t r o n g b i n d i n g s i t e i s no l o n g e r p r e s e n t , s u g g e s t i n g t h a t t h e Asp 153 and Asp 71 a r e no l o n g e r p r o x i m a l ; i n a d d i t i o n , i t i s now p o s s i b l e t h a t t h e a d d i t i o n a l r e s i d u e s Asp 90, Asp 194, and Asp 189 a r e e x p o s e d , l e a d i n g t o a t o t a l o f somewhere between 4 and 7 s i t e s f o r weak b i n d i n g o f M n + + . Keepi n g i n mind t h a t t h e o b s e r v e d r e s u l t s w i l l * see BMD-X85 non l i n e a r l e a s t s q u a r e f i t , c o mputing c e n t e r , U. B. C. - 44 -r e p r e s e n t t h e sum o f a c t i v e and i n a c t i v e t r y p s i n , , i t seems l i k e l y t h a t t h e a v e r a g e number o f weak b i n d i n g s i t e s w i l l be somewhere between 3=((2+4)/2) and 4.5=((2+7)/2). The computer was t h u s asked t o s e a r c h f o r f i t s u s i n g v a l u e s o f 3 < n 2 < 4 . 5 . So, t r y p s i n p o s s e s s e s one s t r o n g b i n d i n g s i t e , w i t h = 2900 l i t e r m o l e ~ \ and s e v e r a l ( n g ' M . S ) weak b i n d i n g s i t e s o f a v e r a g e b i n d i n g c o n s t a n t . K 2 =53 l i t e r m o l e - ^ . At v e r y h i g h [ M n + + ] l e v e l s even more b i n d i n g s i t e s may be p o p u l a t e d , as shown by t h e p o s i t i o n o f t h e r i g h t - m o s t p o i n t i n F i g u r e 2.2. 2. R o t a t i o n a l l a b i l i t y o f water a t t h e s t r o n g M n + + - b i n d i n g  s i t e o f t y r p s i n . A l t h o u g h eq. (2.6) i s e x p e c t e d t o a p p l y t o t h e p r e s e n t e x p e r i m e n t s , t h e d a t a was a n a l y z e d a c c o r d i n g t o t h e more g e n e r a l eq. (2.7) as f o l l o w s . In a s e t o f c o n t r o l e x p e r i m e n t s , T-j and T 2 were measured f o r s o l u t i o n s c o n t a i n i n g b u f f e r and M n + + but no enzyme: t h e a p p r o p r i a t e f o r m f o r T-| i s g i v e n by WVobs = ( 1 / T 1 A ) + [ M n + + ] ' 6 • <2-10> ' 0 b S 1 ( T 1 B + x B ) - 5 5 . 6 - 45 -where 6 i s t h e h y d r a t i o n number f o r water around M n + + . In a second c o n t r o l e x p e r i m e n t , enzyme was added t o b u f f e r s o l u t i o n ++ w i t h no Mn p r e s e n t . W h i l e t h e s o l u t i o n v i s c o s i t y i n c r e a s e d somewhat, as shown by t h e l ^ - i n c r e a s e , t h e e f f e c t on was n e g l i g i b l e . The p r i n c i p a l measurements o f T^ and T 2 were t h e n c a r r i e d o u t on s o l u t i o n s which c o n t a i n e d c o n s t a n t amounts o f b u f f e r and enzyme, w i t h v a r y i n g c o n c e n t r a t i o n s o f M n + + ( s e e T a b l e 2 . 1 ) . A c c o r d i n g t o t h e e q u a t i o n , A {[Mnl - [ M n : t r y p s i n ] } ' 6 ( 1 / V * o b s = ( 1 / T i > + —h 1 0 b S 1 (T-| + Tg) *55.6 / [ M n : t r y p s i n ] \ / q \ + ( ) ' [ — ) , (2.11) \ 55.6 / YT, + x c / where q i s t h e number o f w a t e r m o l e c u l e s c o o r d i n a t e d t o bound Mn S u b t r a c t i n g eq. (2.10) from e q. (2.11) t h e n y i e l d s s ++ r [ M n : t r y p s i n ] ) r . .„ . . . . c. . 0 / T . ) * . - d / T , ) . = - — — — T - J — - * — 1 i V o b s l ' o b s j 55.6 S L 7 ^ T 7 c T l B + x B - i (2.12) An e q u a t i o n o f t h e same form i s v a l i d f o r T 2 a l s o . - 46 -Table 2.1 Experimental proton relaxation rates (sec-^) for water protons in 0.05M tris-maleate buffer, pH 7.1, in the presence (*) or absence ( ) of trypsin (12 mg in 0.5 ml), for the concentrations of [Mn++] l isted. Relaxation rates are the average of at least three runs. Solution ( 1 / Tl>obs W V o b s ^'VobS ^ V o b s A [Buffer only] 0.38 0.38 1.2 1.9 A + 10"5M [Mn++] 0.42 0.45 2.09 1.94 A + 10"4M [Mn++] 1.04 1.11 8.8 6.3 A + 10"3M [Mn++] 6.8 7.35 75.0 62.0 A + 10"2M [Mn"*"1"] 64.5 67.0 735.0 690.0 - 47 -Now t h e l e f t - h a n d - s i d e o f eq. (2.12) i s o b t a i n e d from e x p e r i -ment, and t h e second term on the r i g h t - h a n d - s i d e i s o b t a i n e d from c o r r e s p o n d i n g c o n t r o l e x p e r i m e n t s . A v a l u e f o r t h e b i n d i n g c o n s t a n t f o r M n + + t o t r y p s i n i s now g u e s s e d , and t h e computer p e r f o r m s a n o n - l i n e a r l e a s t - s q u a r e s f i t o f eq. (2.12) t o e x p e r i -mental T ^ - d a t a , and l i s t s t h e s t a n d a r d d e v i a t i o n f o r t h e c h o i c e o f [ q / ( T 2 + T^.] which gave t h e b e s t agreement. The p r o c e d u r e i s t hen r e p e a t e d f o r a r a n g e o f o t h e r v a l u e s f o r t h e b i n d i n g c o n s t a n t , and t h e b e s t f i t was f o u n d f o r (1/K) = 1.3 ± 1.0 x 10" 3M. Due t o t h e c o n t a c t i n t e r a c t i o n i n v o l v e d i n t h e r e l a x a t i o n p r o c e s s f o r Tg, no f u r t h e r use was made o f T 2 - d a t a i n a n a l y s i s o f m o l e c u l a r m o t i o n o f w a t e r . T-j d a t a can be t r e a t e d t h e same way t o g i v e t h e terms q / ( T 1 M + T C ) and q / ( T 2 M + x c ) . By t r e a t i n g t h e d a t a t h i s way one w i l l have a b e t t e r e s t i m a t e o f t h e e x a c t p a r a m a g n e t i c c o n t r i -b u t i o n t o t h e s p i n - l a t t i c e r e l a x a t i o n t i m e and s p i n - s p i n r e l a x a -t i o n t i m e , t h a n t h a t from d i s c u s s i o n based i n terms o f enhancement f a c t o r , e. To t e s t t h e c o n s i s t e n c y o f t h e a p p r o a c h , a s e c o n d s e t o f computer f i t s was p e r f o r m e d w i t h t h e a s s u m p t i o n t h a t t h e s t o i c h i o -metry o f t h e M n r t r y p s i n complex was 2:1 r a t h e r t h a n 1:1. These f i t s showed t e n t i m e s l a r g e r s t a n d a r d d e v i a t i o n ( p o o r e r f i t s ) t h a n f o r t h e 1:1 a s s u m p t i o n . T h u s , t h e NMR r e l a x a t i o n d a t a do p r o v i d e a c c u r a t e s t o i c h i o m e t r y , but t h e b i n d i n g c o n s t a n t i s o b t a i n e d much more r e l i a b l y from EPR peak h e i g h t d a t a , - 48 -i n c o n t r a s t t o t h e example quoted by Dwek , u. The n e x t s t e p i s t o d e t e r m i n e whether T ^ M o r i s t h e main c o n t r i b u t o r f o r t h e l o n g i t u d i n a l r e l a x a t i o n . S i n c e T f o r water C A ++ -7 21 on aqueous Mn i s abo u t 10" s e c , and s i n c e t h e l i f e t i m e f o r wat e r on M n + + i s abo u t t h e same o r even a l i t t l e s h o r t e r i n t h e + + 1 5 p r e s e n c e o f l i g a t i o n o f t h e Mn , i t seems _ver'y l i k e l y t h a t t h e l i f e t i m e f o r water on t h e M n : t r y p s i n complex s h o u l d be o f t h e o r d e r o f 1 0 ~ 7 s e c . Thus, i f t h e d e n o m i n a t o r o f t h e f i r s t t e r m o f e q . (2.12) were dominated by x c , t h a t term s h o u l d be o f t h e o r d e r o f 10^. S i n c e t h e d e t e r m i n e d magnitude o f t h a t term 4 i s o n l y a b o u t 10 , we can be c o n f i d e n t t h a t t h e term i s dominated C by T.j , n o t by r^. We a r e now i n a p o s i t i o n t o d i s c u s s r o t a t i o n a l C m o t i o n o f w a t e r f r o m a n a l y s i s o f . Wit h 6/(1^ 8 + x g ) = 3,56 x 1 0 5 o b t a i n e d f r o m t h e l e a s t s q u a r e f i t . o f t h e d a t a w i t h o u t enzyme, e q u a t i o n (2.12) can be w r i t t e n a s (2.13) S i n c e t h e d e t e r m i n a t i o n o f b i n d i n g c o n s t a n t s by EPR i s more a c c u r a t e t h a n by NMR, t h e r e s u l t s o f F i g u r e 2.2 may now be used *N0TE: T c = r e s i d e n c e t i m e a t s i t e C, n o t r o t a t i o n a l c o r r e l a t i o n t i m e i n t h i s c h a p t e r . 4 - 49 -t o o b t a i n a b e t t e r v a l u e f o r (q/T-j ) , p r o v i d e d t h a t t h e c o n t r i -b u t i o n from t h e weak b i n d i n g s i t e s can be t a k e n i n t o a c c o u n t . For t h e NMR d a t a , t h e n , t h e h i g h - [ M n + + ] s o l u t i o n s w i l l e x h i b i t r a s u b s t a n t i a l c o n t r i b u t i o n t o from t h e weak b i n d i n g s i t e s , but th e l o w - [ M n + + ] s o l u t i o n s show such a s m a l l change i n T ^ o b s t h a t r a c c u r a t e c a n n o t be o b t a i n e d . A compromise i s p r o v i d e d by ++ -3 t h e T-j d a t a f o r i n t e r m e d i a t e [Mn ] = 10 M. Even a t t h i s c o n -r c e n t r a t i o n , t h e r e i s some c o n t r i b u t i o n t o from t h e weak b i n d i n g s i t e s : upper and l o w e r l i m i t s f o r t h i s e f f e c t may be fo u n d f r o m s u p p o s i n g t h a t t h e w a t e r a t t h e weak b i n d i n g s i t e i s t h e same as a t f r e e manganese i o n , o r t h a t a t t h e weak b i n d i n g s i t e i s t h e same as a t t h e s t r o n g b i n d i n g s i t e . W ith a l i t t l e a l g e b r a , e q u a t i o n (2.8) and (2.9) can be r e a r r a n g e d t o [Mn]^ = [ M n : t r y p s i n ] t h e f i r s t t erm r e p r e s e n t s t h e number o f M n + + bound a t s t r o n g b i n d i n g s i t e s , t h e second t e r m w i l l be t h e number o f M n + + a t weak b i n d i n g s i t e s . So, t h e term [ M n : t r y p s i n ] i n e q u a t i o n (2.13) w i l l be e i t h e r e q u a l t o t h e f i r s t t erm o n l y o r equ a l t o both terms i n e q u a t i o n (2.14) d e p e n d i n g on whether a t t h e weak b i n d i n g s i t e s i s ,the same as T, a t f r e e manganese, o r T, a t t h e weak b i n d i n g s i t e i s - 50 -t h e same as a t t h e s t r o n g b i n d i n g s i t e . n-j, n 2 > K-|, K 2 have been d e t e r m i n e d by a S c a t c h a r d p l o t o f t h e ESR d a t a ( F i g u r e 2 . 2 ) ; [Mn]p i s t h e m e a s u r a b l e q u a n t i t y f r o m ESR t h u s [ M n : t r y p s i n ] can be c a l c u l a t e d f o r each c a s e by r u s i n g eq. ( 2 . 1 4 ) . q / ( T 1 can be f o u n d by e q u a t i o n (2.13) f r o m [ M n : t r y p s i n ] c a l c u l a t e d f o r t h e s e two e x t r e m e s . The f i n a l d e t e r m i n a t i o n o f T-j f o r t h e s t r o n g b i n d i n g s i t e i s t h e n o b t a i n e d from t h e o b s e r v e d T^ as 4.18 x 10° < — r < 4.46 x 10° s e c " 1 (2.15) T l To a n t i c i p a t e what w i l l s h o r t l y be shown, t h e c o r r e l a t i o n t i m e c a l c u l a t e d f r o m t h e T y C o f (2.15) must be 2.5 x 1 0 ~ 1 0 s e c o r s h o r t e r . S i n c e t h i s c o r r e l a t i o n t i m e i s g i v e n by ( l / x c ) = ( l / T r o t ) + ( l / x s ) , (2 .16) -9 22 and s i n c e T i s 2 x 10 s e c , i t i s e v i d e n t t h a t x = x ., s c r o t and w i l l be so i n t e r p r e t e d f r o m h e r e on. B.M. Fung has r e c e n t l y c a l c u l a t e d t h e e f f e c t o f i n t e r n a l 23 r o t a t i o n on t h e e l e c t r o n - n u c l e a r d i p o l e - d i p o l e i n t e r a c t i o n , w i t h r e s u l t s t h a t c l o s e l y r e s e m b l e an e a r l i e r c a l c u l a t i o n f o r the 24 n u c l e a r - n u c l e a r d i p o l e - d i p o l e c a s e : 1 _ JVYS^SCS + I ) ) ( 7 r ( 3 c o s 2 9 ) 2 T l 3 ( s i n 2 2 9 ) x 2 3 ( s i n 4 e ) x . T 7 - ) T r ) ( 30 L I +V T l Z 1 + W S 2 t 2 2 1 + W S 2 T 3 2 ' - 51 -.1 r ( 3 c o s 2 e - l ) 2 T l 3 ( s i n 2 2 e K 2 3 ( s i n 4 e ) T 3 10 L 1 + W l 2 X l 2 1 +Wj 2T 2 3 1 +W I 2T 3 2-< , (2.17) where (1/T-,) = ( I / O i n t h i s c a s e , and (1/TJ = (1/T,) + (1/T ) ' r L 1 r i n t and (1/TO) = (1/T,) + (4/T ), where T i s t h e r o t a t i o n a l 13 1 r i n t r c o r r e l a t i o n t i m e f o r t h e complex as a whole and T i s t h e c o r r e l a t i o n t i m e f o r i n t e r n a l r o t a t i o n o f w a t e r i n t h e complex, and e i s t h e a n g l e between t h e p r o t o n - p r o t o n v e c t o r i n water and t h e a x i s about which i n t e r n a l r o t a t i o n o c c u r s . F o r example, f o r M n ( H 2 0 ) 6 + + ( F i g u r e 2 . 3 ) , e = 9 0 ° f o r t h e most l i k e l y i n t e r n a l r o t a t i o n a x i s t h r o u g h t h e oxygen atom, and i f t h e p r e v i o u s l y c a l c u l a t e d c o r r e l a t i o n t i m e f o r w a t e r r o t a t i o n i s t a k e n as -11 25 3 x 10 = . th a n one o b t a i n s from t h e p r e s e n t d a t a f o r r m t B 4 - 1 (1 /T-j ) = 5.9 x 10 s e c , t h a t t h e r o t a t i o n a l c o r r e l a t i o n t i m e f o r t h e M n ( H 2 0 ) 6 + + complex a s a whole i s 4.8 x 1 0 " ^ s e c = T^, as shown g r a p h i c a l l y i n F i g u r e 2.4. F o r t h e M n t t r y p s i n complex, assuming t h a t t h e bound w a t e r r o t a t e s f r e e l y ( T„. ^  = 3 x 1 0 " ^ r m t s e c ) , t h e f i g u r e shows t h a t t h e c o r r e l a t i o n t i m e f o r r o t a t i o n o f t h e M n - b i n d i n g s i t e i s 8 x 1 0 " ^ s e c . I f t h e bound w a t e r i s assumed r i g i d l y h e l d t o t h e M n r t r y p s i n s i t e , t h e n t he b i n d i n g s i t e i s even more f l e x i b l e . T h e s e c a l c u l a t i o n s a r e ba s e d on i a s s u m p t i o n o f q = 4 water m o l e c u l e s p e r bound M n + + . I f q i s ta k e n as 2, o r 3, t h e n t h e c o r r e l a t i o n t i m e f o r r o t a t i o n o f the Mn - b i n d i n g s i t e w i l l be 2.5 x 10"^° o r 1.4 x 10"^° r e s p e c t i v e l y . T h u s , t h e M n + + bound t o t r y p s i n has a s u r p r i s i n g l y l a r g e d e g r e e -52-F i g u r e 2.3 The p o s s i b l e s t r u c t u r e o f Mn s t r o n g b i n d i n g s i t e on T r y p s i n . The a n g l e between the a x i s o f i n t e r n a l r o t a t i o n and t h e a x i s c o n n e c t i n g the two p r o t o n s o f w a t e r i s s e e n t o be 9 0 ° . Here i t i s assumed t h a t two w a t e r m o l e c u l e s a r e r e p l a c e d by two A s p a r t i c r e s i d u e s o f t r y p s i n ( s e e t e x t ) . - 53 -- 54 -F i g u r e 2 . 4 L o g - l o g p l o t o f l o n g i t u d i n a l r e l a x a t i o n r a t e v e r s u s m a c r o m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n t i m e , u s i n g eq. ( 2 , 1 7 ) o f t h e t e x t . R e l a x a t i o n r a t e s a r e c a l c u l a t e d f o r a wa t e r m o l e c u l e c o o r d i n a t e d t o ++ ++ °27 Mn , w i t h proton-Mn d i s t a n c e o f 2 . 8 A , 8 - 1 toj = 2ir x 10 s e c " , and i n t e r n a l r o t a t i o n r a t e f i x e d by T = 3 x 1 0 - 1 1 s e c . ( b o t t o m c u r v e ) . The r i n t upper c u r v e r e p r e s e n t s t h e same c a l c u l a t i o n , but w i t h no i n t e r n a l r o t a t i o n p r e s e n t , t h a t i s T.. ^ ->• » i n eq. ( 2 , 1 7 ) . S o l i d - l i n e i n t e r c e p t s r e f e r t o (1 /T-j) and x 1 f o r t h e M n ( H 2 0 ) g + + complex; d o t t e d - l i n e i n t e r c e p t s a r e f o r w a ter bound t o t h e M n : t r y p s i n complex ( s e e D i s c u s s i o n ) . - 55 -&L/l)5ol - 56 -o f r o t a t i o n a l m o b i l i t y : i f t h e M n T T were r i g i d l y bound t o t h e t r y p s i n "backbone", i t s r o t a t i o n a l c o r r e l a t i o n t i m e would be about two o r d e r s o f magnitude s l o w e r . ++ Most p r e v i o u s s t u d i e s on Mn - i n d u c e d w a t e r n u c l e a r r e l a x a -t i o n have been based on r a t i o s o f r e l a x a t i o n r a t e s o r "enhancements" r a t h e r t h a n on d i f f e r e n c e s i n r e l a x a t i o n r a t e s as i n the p r e s e n t c a s e . As d e m o n s t r a t e d r e c e n t l y f o r n u c l e a r - n u c l e a r d i p o l e - d i p o l e 26 r e l a x a t i o n , whenever i n t e r n a l m o t i o n i s p r e s e n t , t h e i n f o r m a t i o n o b t a i n e d f r o m r a t i o s o f r e l a x a t i o n t i m e s o f f e r s a q u e s t i o n a b l e m i x t u r e o f t h e r o t a t i o n a l r a t e o f t h e m a c r o m o l e c u l e s a s a whole and t h e i n t e r n a l r o t a t i o n a l r a t e . S i n c e t h e o n l y c a s e s f o r which Mn - i n d u c e d p r o t o n r e l a x a t i o n o f f e r s r o t a t i o n a l i n f o r m a t i o n a r e when t h e r o t a t i o n a l " e f f e c t i v e " c o r r e l a t i o n t i m e i s s h o r t e r -9 t h a n t h e e l e c t r o n o f a b o u t 2 x 1 0 s e c , and s i n c e t y p i c a l -9 enzyme r o t a t i o n a l c o r r e l a t i o n t i m e s a r e 10 - 100 x 10 s e c , i t i s c l e a r t h a t t h e t y p i c a l s i t u a t i o n i n w h i c h M n + + i s a u s e f u l n u c l e a r m a g n e t i c probe o f enzyme l o c a l m o t i o n i s one f o r w hich i n t e r n a l r o t a t i o n i s s i g n i f i c a n t l y f a s t e r t h a n m a c r o m o l e c u l a r r e o r i e n t a t i o n as a whole. We, t h e r e f o r e , s u g g e s t t h a t f u t u r e NMR r e l a x a t i o n s t u d i e s o f water i n t h e p r e s e n c e o f M n + + complexes w i t h m a c r o m o l e c u l e s s h o u l d base t h e i r i n t e r p r e t a i t o n s on d i f f e r -ence i n r e l a x a t i o n r a t e i n p r e s e n c e and a b s e n c e o f enzyme, r a t h e r t h a n on r a t i o s , o r "enhancements" o f r e l a x a t i o n r a t e s . - 57 -E. Summary The s t r e n g t h and number o f b i n d i n g s i t e s o f M n + + t o t r y p s i n have been d e t e r m i n e d by a S c a t c h a r d r e d u c t i o n o f e l e c t r o n p a r a -m a g n e t i c r e s o n a n c e peak h e i g h t d a t a . A c t i v e t r y p s i n p o s s e s s e s one s t r o n g b i n d i n g s i t e w i t h b i n d i n g c o n s t a n t 2900 1 mole"^; t h e r e a r e 4-5 b i n d i n g s i t e s w i t h a much weaker b i n d i n g c o n s t a n t o f 53 1 mole"^. B i n d i n g seems most l i k e l y t o i n v o l v e c o o r d i n a t i o n t o Asp 153 and Asp 71. I n a c t i v e t r y p s i n has no s t r o n g M n - -b i n d i n g s i t e . F l e x i b i l i t y a t t h e b i n d i n g s i t e has been d e t e r m i n e d f r o m a n a l y s i s o f p r o t o n l o n g i t u d i n a l m a g n e t i c r e l a x a t i o n r a t e s f o r w a t er a t t h e s t r o n g M n + + - b i n d i n g s i t e . U s i n g d i f f e r e n c e s ( r a t h e r t h a n r a t i o s ) between r e l a x a t i o n r a t e s i n p r e s e n c e and a b s e n c e o f enzyme, t h e f l e x i b i l i t y o f t h e b i n d i n g s i t e i t s e l f i s d e t e r m i n e d t o have a r o t a t i o n a l c o r r e l a t i o n t i m e o f 8 x 1 0 " ^ s e c o r s h o r t e r , so t h a t t h e r e i s a p p r e c i a b l e l o c a l m o t i o n a t t h e ++ s t r o n g Mn - b i n d i n g s i t e . - 58 -R e f e r e n c e s 1. A.S. M i l d v a n and M. Cohn, Advan. Enzymol. 33, 1 ( 1 9 7 0 ) . 2. J . E i s i n g e r , R.G. Shultnan and B.M. Sz y m a n s k i , J . Chem. Phys. 36, 1721 (196 2 ) . 3. L. G o r i n i , B i o c h i m . e t B i o p h y s . A c t a 7_, 318 (1 9 5 1 ) ; M. B i e r and F.F. Nord. A r c h . Biochem. and B i o p h y s . ' 3 3 , 320 (1951) 4. M. Delaage and M. L a z d u n s k i , B i o c h i m . e t B i o p h y s . A c t a 105, 532 ( 1 9 6 5 ) . 5. M.R. McDonald and M. K u n i t z , J . Gen. P h y s i o l . 25_, 53 ( 1 9 4 1 ) . 6. N.M. Green and H. Meurath, J . B i o l . Chem. 204, 379 ( 1 9 5 3 ) . 7. D.W. D a r n a l l and E.R. Birnbaum, J . B i o l . Chem. 245, 6484 (1970), 8. D.A. D e r a n l e a u , J . Amer. Chem. Soc. 91_, 4044, 4050 ( 1 9 6 9 ) . 9. J . T . E d s a l l and J . Wyman, B i o p h y s i c a l C h e m i s t r y , V o l . 1, Academic P r e s s , N.Y., ( 1 9 5 8 ) , pp. 610-618. 10. R.A. Dwek, i n Advances i n M o l e c u l a r R e l a x a t i o n P r o c e s s e s , W.J. O r v i l i e - T h o m a s e d . , E l s e v i e r P u b l i s h i n g Company, Amsterdam, V o l . 4, pp. 1-53 ( 1 9 7 2 ) . 11. T . J . S w i f t and R.E. C o n n i c k , J . Chem. Phys. 37_, 307 ( 1 9 6 2 ) . 12. H.A. Degani and D. F i a t , J . Amer. Chem. Soc. 93_, 4281 ( 1 9 7 1 ) . 13. A. Danchin and M. Gueron, J . Chem. Phys. 53, 3599 ( 1 9 7 0 ) . 14. H.M. M c C o n n e l l , J . Chem. Phys. 28, 430 ( 1 9 5 8 ) . 15. J.P. Hunt, C o o r d . Chem. Rev. 7_, 1 ( 1 9 7 1 ) . 16. T. Chase, J r . , and E. Shaw, Biochem. B i o p h y s . Res. Commun. 29, 508 ( 1 9 6 7 ) . - 59 -17. N. Bloembergen, E.M. P u r c e l l , and R.V. Pound, Phys. Rev. 73, 679 ( 1 9 4 8 ) . 18. S. Meiboom and D. G i l l , Rev. S c i . I n s t r u m . 29_, 688 ( 1 9 5 8 ) . 19. M. Cohn and J . Townsend, Nature'173_» 1090 ( 1 9 5 4 ) . 20. R.M. S t r o u d , L.M. Kay, and R.E. D i c k e r s o n , C o l d S p r i n g H a r b o r Symposium on Q u a n t i t a t i v e B i o l o g y 36_, 125 ( 1 9 7 1 ) . 21. M. G r a n t , H.W. Dodgen and J.P. Hunt, I n o r g , Chem. 1_0_, 71 (1 9 7 1 ) . 22. M. R u b e n s t e i n , A. Baram, and Z. Luz. M o l . Phys. 20, 67 ( 1 9 7 1 ) . 23. B.M. Fung, J . Chem. Phys. 58, 192 ( 1 9 7 3 ) . 24. A.G. M a r s h a l l , P.G. Sc h m i d t and B.D. S y k e s , B i o c h e m i s t r y 11_, 3875 ( 1 9 7 2 ) . 25. N. Bloembergen and L.O. Morgan, J . Chem. Phys. 34, 842 ( 1 9 6 1 ) . 26. L.G. Werbelow and A.G. M a r s h a l l , J . Amer. Chem. Soc..95,5132 ( 1 9 7 3 ) . 27. L.O. Morgan and A.W. N o l l e , J . Chem. Phys. 31, 365 ( 1 9 5 9 ) . - 60 -CHAPTER I I I S t e a d y - S t a t e I n h i b i t i o n K i n e t i c s U s i n g Racemic S u b s t r a t e : A Probe F o r C o o p e r a t i v e I n h i b i t o r B i n d i n g In T r y p s i n A. I n t r o d u c t i o n A c c u m u l a t i n g e v i d e n c e f r o m x - r a y c r y s t a l l o g r a p h y o f c h y m o t r y p s i n and o t h e r enzymes i n d i c a t e s t h a t many enzymes p o s s e s s one o r more s e c o n d a r y b i n d i n g s i t e s which w i l l a t t r a c t v a r i o u s i n h i b i t o r s t o t h e enzyme. W h i l e t h e s e s i t e s i n c h y m o t r y p s i n a r e p r o b a b l y an e v o l u t i o n a r y c o i n c i d e n c e c o n n e c t e d t o h o m o l o g i e s i n s t r u c t u r e w i t h o t h e r s e r i n e e s t e r a s e s , s e c o n d a r y b i n d i n g s i t e s have c e n t r a l 2 s i g n i f i c a n c e i n t h e mechanism o f o p e r a t i o n o f a l l o s t e r i c enzymes . In s o l u t i o n , an enzyme i n h i b i t o r i s most o f t e n c a t e g o r i z e d as " c o m p e t i t i v e " , " n o n - c o m p e t i t i v e " , e t c . , by t h e f o r m o f a D i x o n p l o t o f r e c i p r o c a l r e a c t i o n i n i t i a l r a t e , ( 1 / v ) , v e r s u s i n h i b i t o r w h ich shows " c o m p e t i t i v e " i n h i b i t i o n as j u d g e d by a Dixon p l o t i s g e n e r a l l y presumed t o b i n d as t h e same s i t e as t h e n a t i v e s u b s t r a t e , and t h u s "the s t r e n g t h o f b i n d i n g o f s u c h an i n h i b i t o r can be c o r r e l a t e d w i t h t h e shape o f t h e s u b s t r a t e - b i n d i n g s i t e o f t h e enzyme. However, most enzymes a c t on o p t i c a l l y a c t i v e s u b s t r a t e s i n n a t u r e , and i t may be i n c o n v e n i e n t t o s t u d y i n h i b i t i o n by u s i n g - 61 -a p u r e "L" o r "D" o p t i c a l isomer a r t i f i c i a l s u b s t r a t e ; i n s u c h c a s e s , o f w h i c h t r y p s i n i s one, a r a c e m i c s u b s t r a t e i s u s e d . The i n i t i a l c o m p l i c a t i o n here i s t h a t o n l y one o f t h e o p t i c a l i s o m e r s w i l l i n g e n e r a l be a "good" s u b s t r a t e , and t h e o t h e r may v e r y w e l l be a c o m p e t i t i v e i n h i b i t o r , as w i t h t h e p r e s e n t example. The i m p o r t a n c e o f t h i s f a c t i s t h a t an unknown i n h i b i t o r may a p p e a r t o be " c o m p e t i t i v e " when a s s a y e d by s u c h a r a c e m i c s u b s t r a t e , when i n f a c t t h e unknown i n h i b i t o r does not p r e v e n t b i n d i n g o f s u b s t r a t e . The a p p a r e n t c o m p l i c a t i o n o f a r a c e m i c s u b s t r a t e , however, can be t u r n e d t o a d v a n t a g e , t o p r o v i d e a means f o r c l a s s i f y i n g i n h i b i t o r s a c c o r d i n g t o whether t h e y a r e p u r e l y c o m p e t i t i v e w i t h s u b s t r a t e , r e p u l s i v e l y i n t e r a c t w i t h s u b s t r a t e , a r e p u r e l y non-c o m p e t i t i v e (have no e f f e c t on s u b s t r a t e b i n d i n g ) , o r a c t i v a t e s u b s t r a t e b i n d i n g . I t s h o u l d be n o t e d t h a t no a d d i t i o n a l e x p e r i m e n t a l d a t a beyond t h e o r d i n a r y D i x o n p l o t i s r e q u i r e d i n t h i s a n a l y s i s . In t h i s c h a p t e r t h e above c o n s i d e r a t i o n s a r e a p p l i e d t o t h e a n a l y s i s o f s e v e r a l a r g i n i n e o r l y s i n e a n a l o g s w hich a r e i n h i b i t o r s o f t r y p s i n , and i t i s a l s o shown t h a t some o t h e r i n h i b i t o r s p r e v i o u s l y c l a s s i f i e d as " c o m p e t i t i v e " do n o t i n f a c t p r e v e n t b i n d i n g o f s u b s t r a t e and t h u s must b i n d e l s e w h e r e t h a n a t t h e s u b s t r a t e " p o c k e t " . T r y p s i n shows a v e r y narrow s p e c i f i c i t y f o r c a t a l y z i n g t h e h y d r o l y s i s o f bonds i n v o l v i n g t h e c a r b o x y l group o f a r g i n i n e o r 4 5 l y s i n e * . A s e r i e s o f g u a n i d i n e o r l y s i n e a n a l o g s w h i c h e a c h p o s s e s s e d a -CH^ o r -OCH^ group was c o l l e c t e d and/or s y n t h e s i z e d t o p r o v i d e f o r s i m p l e i n t e r p r e t a t i o n o f NMR r e s u l t s as w e l l as - 62 -c o n v e n t i o n a l s t e a d y - s t a t e i n h i b i t i o n k i n e t i c s . The U.V. s t u d i e s r e p o r t e d h e r e w i l l p r o v i d e t h e n e c e s s a r y i n f o r m a t i o n t o e s t a b l i s h where t h e i n h i b i t o r b i n d s t o t h e enzyme, and t h u s f a c i l i t a t e t h e i n t e r p r e t a t i o n o f t h e NMR e x p e r i m e n t i n the n e x t c h a p t e r . T h i s system would o f f e r f o r t h e f i r s t t i me a c o m p a r i s o n between t h e s t r e n g t h o f b i n d i n g ( f r o m i n h i b i t i o n k i n e t i c s ) and r i g i d i t y o f b i n d i n g ( f r o m NMR), and t h e t r y p s i n system was c h o s e n f o r i t s s p e c i f i c i t y t o a s s u r e t h a t a l l t h e i n h i b i t o r s s h o u l d b i n d c o m p e t i t i v e l y a t t h e a c t i v e s i t e . A l t h o u g h no X - r a y e v i d e n c e f o r s e c o n d a r y b i n d i n g s i t e s on t r y p s i n has y e t a p p e a r e d , a v a r i e t y o f o t h e r work ( b e s i d e s t h e p r e s e n t e x p e r i m e n t s ) p o i n t s t o a d d i t i o n a l b i n d i n g . I t has been o b s e r v e d t h a t t h e s u b s t r a t e , T A M E . ( p - t o l u e n e s u l f o n y l a r g i n i n e methyl e s t e r ) i s s e l f - a c t i v a t i n g a t h i g h c o n c e n t r a t i o n s ^ , s u g g e s t i v e o f an a u x i l i a r y b i n d i n g s i t e . More r e c e n t l y i t has been r e p o r t e d t h a t even n e u t r a l compounds^ can b i n d t o t r y p s i n , r e s u l t i n g i n c o o p e r a t i v e i n t e r a c t i o n w i t h s u b s t r a t e . A l t h o u g h M a r e s - G u i a and Shaw r e p o r t e d t h a t a l l seven o f t h e i n h i b i t o r s t h e y s t u d i e d were c o m p e t i t i v e , a r e - a n a l y s i s o f t h e i r r e s u l t s ( s e e D i s c u s s i o n ) shows t h a t two o f t h e s e were not t r u l y c o m p e t i t i v e , b ut a c t e d t o r e s t r i c t , r a t h e r t h a n p r e v e n t a c c e s s o f s u b s t r a t e t o i t s b i n d i n g s i t e . F i n a l l y , s i n c e C a + + i s known t o s t a b i l i z e t r y p s i n 9 10 a g a i n s t a u t o l y s i s , and a l s o a g a i n s t a c i d , b a s e , and u r e a , c a l c i u m i o n s was p r e s e n t i n a l l a s s a y s , and i t s e f f e c t w i l l be d i s c u s s e d i n t h e R e s u l t s s e c t i o n . - 63 -B. E x p e r i m e n t a l B o v i n e t r y p s i n , t w i c e - r e c r y s t a l l i z e d , s a l t - f r e e , l y o p h i l i z e d p r e p a r a t i o n (TRL) from W o r t h i n g t o n B i o c h e m i c a l C o r p . , F r e e h o l d , N.J., was used w i t h o u t f u r t h e r p u r i f i c a t i o n . O t h e r compounds were examined by NMR f o r i m p u r i t y p e a k s . 3-methoxypropylamine ( E a s t e r n O r g a n i c C h e m i c a l s ) . The t e c h n i c a l g r a d e compound was s t i r r e d o v e r n i g h t w i t h sodium c a r b o n a t e and f r a c t i o n a l l y d i s t i l l e d a f t e r w a r d s . B.P. 116-117°C. A c e t a m i d i n e - H C l ( J . T . Baker C h e m i c a l Co.) was r e c r y s t a l l i z e d t w i c e from e t h a n o l t o g i v e a w h i t e powder, m.p. 174°C. p-methoxybenzylami ne ( J . T . Baker C h e m i c a l Co.) was used w i t h o u t f u r t h e r p u r i f i c a t i o n . m-anisidine»HCl (Eastman O r g a n i c C h e m i c a l s ) was c r y s t a l l i z e d t w i c e f r o m a b s o l u t e e t h a n o l t o g i v e a w h i t e powder, m.p. 176-178°C. p - a n i s i d i n e ( E a s t e r n O r g a n i c C h e m i c a l s ) was c r y s t a l l i z e d t w i c e f r o m w a t e r t o g i v e l i g h t brown p l a t e s , m.p. 57°C. m - m e t h o x y p h e n y l g u a n i d i n e h y d r o c h l o r i d e was s y n t h e s i z e d by a m o d i f i c a t i o n o f a p r e v i o u s m e t h o d 1 1 . l - g u a n y l - 3 , 5 - d i m e t h y l p y r a z o l e n i t r a t e ( 8 g , 0.04 mo l e s ) and m - a n i s i d i n e (50g, 0.4 mo l e s ) were mixed i n 160 ml h^O, and t h e s o l u t i o n r e f l u x e d u n t i l t h e GDPN had d i s a p p e a r e d ( a s gauged by i t s NMR s p e c t r u m ) . The m i x t u r e was a l l o w e d t o c o o l and t h e n e x t r a c t e d e i g h t t i m e s w i t h 40 c c p o r t i o n s o f e t h e r . The acqueous l a y e r was d e c o l o r i z e d w i t h a c t i v e c a r b o n , and t h e n put t h r o u g h a Dowex 2-X8 i o n exhange r e s i n ( C l - form) t o c o n v e r t t h e n i t r a t e s a l t t o a c h l o r i d e s a l t . A f t e r f r e e z e -- 64 -d r y i n g , t h e s o l i d s were d i s s o l v e d i n a s m a l l amount o f e t h a n o l , 12 and p a s s e d t h r o u g h a p r e c o n d i t i o n e d s i l i c a g e l column (100 mesh), e l u t e d w i t h b e n z e n e : e t h a n o l m i x t u r e s v a r y i n g f r o m 4:1 t o 1:4 c o m p o s i t i o n . The g u a n i d i n e p r o d u c t was i d e n t i f i e d by S a k a g u c h i 13 14 t e s t and a l s o Weber t e s t . Tubes w i t h p o s i t i v e r e a c t i o n s were p o o l e d , s o l v e n t e v a p o r a t e d under vacuum, t h e n d i s s o l v e d i n a b s o l u t e e t h a n o l , f o l l o w e d by a d d i t i o n o f a few d r o p s o f c o n c e n t r a t e d a c e t i c a c i d and t h e n e t h y l a c e t a t e u n t i l p r e c i p i t a t i o n was i n i t i a t e d . The s o l u t i o n was t h e n s t o r e d i n a f r e e z e r o v e r n i g h t t o g i v e a maximum y i e l d o f p r e c i p i t a t e , i d e n t i f i e d by NMR. A p o r t i o n o f t h e m-methoxyphenyl-g u a n i d i n e h y d r o c h l o r i d e was c o n v e r t e d t o t h e base by a d d i t i o n o f NaOH i n i c e c o l d w a t e r , f o l l o w e d by e x t r a c t i o n w i t h e t h e r and e v a p o r a t i o n o f t h e e t h e r w i t h a w a t e r pump. The base was th e n d i s s o l v e d i n C D C l ^ . and NMR gave a r a t i o o f g u a n i d i n e t o methyl p r o t o n s o f 4-5 t o 3. The uv a b s o r p t i o n band o f m - m e t h o x y p h e n y l g u a n i d i n e h y d r o c h l o r i d e was s h i f t e d t o low w a v e l e n g t h compared t o m - a n i s i d i n e i n m e t h a n o l , and t h e uv spe c t r u m was s i m i l a r t o t h e known p h e n y l g u a n i d i n e -H C l . m.p. was 115-116°C and t h i n - l a y e r c h r o m a t o g r a p h y showed no o t h e r i m p u r i t i e s : A n a l y s i s c a l c d . f o r CgH^N-jOCl: C 47.64 H 5.99 N 20.84 Found: C 46.82 H 5.93 N 20.33 p - m e t h o x y p h e n y l g u a n i d i n e • H C l was s y n t h e s i z e d and t e s t e d as f o r t h e meta-compound, and showed m.p. 137 - 1 3 9°C. T h i n - l a y e r c h r o m a t o g r a p h y showed no o t h e r i m p u r i t i e s : - 65 -A n a l y s i s c a l c d . f o r CgH., gNgOCl: C 47.64 H 5.99 N 20.84 Found: C 46.87 H 5.82 N 1 9 , 9 6 . The d i f f i c u l t i e s i n t h e above s y n t h e s e s l i e i n t h e i n s t a b i l i t y o f t h e p r o d u c t ( h y d r o l y s i s a t h i g h t e m p e r a t u r e o r b a s i c s o l u t i o n ) , and because a s m a l l amount o f i m p u r i t y ( p o s s i b l y b i g u a n i d i n e ) seems t o p r e v e n t c r y s t a l l i z a t i o n . T r y p s i n a s s a y s pH o f a l l b u f f e r s was measured w i t h an O r i o n Model 401 pH m e t e r ; a G i l f o r d uv s p e c t r o p h o t o m e t e r w i t h r e c o r d e r was employed f o r a b s o r b a n c e measurements, and t e m p e r a t u r e was c o n t r o l l e d by a Haake c i r c u l a t o r - t h e r m o s t a t . Enzyme s o l u t i o n was p r e p a r e d by d i s s o l v i n g 2 mg t r y p s i n i n a 5 ml v o l u m e t r i c f l a s k which c o n t a i n e d 0.1 ml o f 0.25M C a C l 2 and t h e r e m a i n d e r c f 0.001N i c e - c o l d H C l . S u b s t r a t e s o l u t i o n s , D , L - b e n z o y l a r g i n i n e - p - n i t r o a n i l i d e -4 -4 -4 [D.L-BAPA], 1 x 10 M, 2x10 M, and 3 x 10 M, were p r e p a r e d f r e s h e v e r y t i m e , because sometimes even a t low c o n c e n t r a t i o n , D,L-BAPA would s t a r t t o c r y s t a l l i z e a t t h e bottom o f t h e v o l u m e t r i c f l a s k ( 3 0 ° C ) . I n h i b i t o r s were p r e p a r e d a t c o n c e n t r a t i o n s a p p r o p r i a t e t o t h e i r p o t e n c y f o r i n h i b i t i o n , b u t u s u a l l y a b o u t 0.05M i n 0.05M t r i s b u f f e r , pH 7.65, w i t h 0.02M CaCl,,. S p e c t r o p h o t o m e t r y a s s a y s 15 were c o n d u c t e d w i t h D,L-BAPA as s u b s t r a t e . T r y p t i c a c t i v i t y was measured by a d d i n g 0.1 ml o f t r y p s i n s o l u t i o n (4 mg i n 10.00 cc o f 0.001N HCl) a t z e r o t i m e t o 2.5 c c o f s u b s t r a t e and 0.4 c c b u f f e r [ o r 2.5 c c s u b s t r a t e p l u s x c c i n h i b i t o r and (0.4 - x) c c b u f f e r ] i n t h e c u v e t t e , w i t h p r i o r t h e r m a l e q u i l i b r a t i o n i n the c e l l compartment o f t h e s p e c t r o p h o t o m e t e r ( 3 0 ° C ) . A f t e r a d d i t i o n o f enzyme, o t h e change i n a b s o r b a n c e a t 4100 A was r e c o r d e d f o r 5 m i n u t e s ; - 66 -i n a l l e x p e r i m e n t s , the r a t e was o b t a i n e d i n t r i p l i c a t e and the r e p o r t e d v a l u e i s an a v e r a g e . The r a t e , i n mole l i t e r " ^ s e c " ^ was o b t a i n e d by m u l t i p l y i n g the measured a b s o r b a n c e p e r m i n u t e by [ 1 / ( 6 0 - 8 8 0 0 ) ] , where 8800 i s t h e molar e x t i n c t i o n c o e f f i c i e n t o o f p - n i t r o a n i l i n e a t 4100 A. C. T h e o r y The p r e s e n t e x p e r i m e n t s i n v o l v e a r a c e m i c s u b s t r a t e whose L-form i s a s u b s t r a t e but whose D-form i s a c o m p e t i t i v e i n h i b i t o r : t h e s e s p e c i e s w i l l be d e n o t e d as S and I , . The r a c e m i c s u b s t r a t e i s t h e n used t o a s s a y an unknown i n h i b i t o r , I 2 , and t h e a p p r o p r i a t e scheme i s shown below: Y K 0 ^ E I 2 S E + P (3,1) where = J H J I L ] [ E J [SJ [ E S ] o l C ] . [EI2] [1,3 CEI1I2] K 2 = [ E ] [ I 2 ] [ E I , ] = [ E I , ] [ I ] • — » [ E ^ L , ] [ E I 2 S ] [EIoS] ( 3 . 2 ) - 67 -I f a = °°, t h e n Ig i s p u r e c o m p e t i t i v e w i t h r e s p e c t t o 1^ ; if y = 00> t h e n i s p u r e c o m p t i t i v e w i t h S. a = 1 and y = 1 a r e t h e c o r r e s p o n d i n g p u r e n o n - c o m p e t i t i v e c a s e s , and so f o r t h . The g e n e r a l s t e a d y - s t a t e s o l u t i o n f o r t h e e f f e c t o f two i n h i b i t o r s 3 on an e n z y m e - s u b s t r a t e s y s t e m i s known ; w i t h t h e p r e s e n t s p e c i a l c a s e , t h e r e s u l t a n t g e n e r a l r a t e e x p r e s s i o n can be s i m p l i f i e d as f o l l o w s . ( l / v ) = h l ( 1 / [ S ] ) * ( [ I l ] / K l [ S ] ) * 0 / K s ) max +. £Izl ((i/[s]) + ([1,3/aK^s]) .+ ( V Y K S ) ) | , (3.3) where v i s the i n i t i a l r a t e o f f o r m a t i o n o f p r o d u c t a t t h e i n i t i a l c o n c e n t r a t i o n s o f S, I , , and I 0 f o r t h e r u n , V i s t h e 1 2 max maximum i n i t i a l r a t e f o r t h a t v a l u e o f I g , and i s g i v e n by t h e u s u a l Vmax - total • <3:4> Now i n t h e p r e s e n t e x p e r i m e n t s , r a c e m i c s u b s t r a t e i s used, so t h a t [ I , ] = [ S ] (3.5) a t a l l t i m e s . W i t h t h i s s i m p l i f i c a t i o n , e q . (3.3) may be r e d u c e d t o - 68 -(1/v) = 1 + 1 + 1 + [ I , ] 1 + 1 K 2 L [ S ] aK, Y K S J . (3.6) Thus a D i x o n - t y p e p l o t o f (1/v) v e r s u s [l^l w i l l s t i l l be a s t r a i g h t l i n e a t a g i v e n s u b s t r a t e c o n c e n t r a t i o n . I f t h e e x p e r i m e n t i s r e p e a t e d a t a second ( f i x e d ) s u b s t r a t e l e v e l , t h e n t h e two Dixon p l o t s can r e a d i l y be shown t o i n t e r s e c t a t t h e p o i n t , [ I 9 ] = -K 9 , (Vv) max K, K, 1 + 1 SJJ (3.7) From e q s . (3.7), i t i s c l e a r t h a t t h e D i x o n p l o t s w i l l i n t e r s e c t above t h e x - a x i s when I 2 i s pu r e c o m p e t i t i v e (a,y = 0 0) o r shows r e p u l s i v e i n t e r a c t i o n (1 < a,y < 0 0) w i t h I, and S; t h e i n t e r s e c t i o n w i l l o c c u r on_ t h e x - a x i s when I 2 i s pure n o n - c o m p e t i t i v e (a,y = 1) w i t h I, and S; and t h e i n t e r s e c t i o n w i l l l i e below t h e x - a x i s when I 2 a c t i v a t e s (a,y < 1) b i n d i n g o f I, and S. Independent e x t r a c t i o n o f t h e i n t e r a c t i o n c o e f f i c i e n t s , a and Y> would r e q u i r e an u n p r o f i t a b l y l a r g e number o f r a t e measurements. However, a p a r a m e t e r o f f e r i n g n e a r l y a s much i n f o r m a t i o n may be d e f i n e d by - 69 -K l KS The p a r a m e t e r , A, p r o v i d e s a w e i g h t e d a v e r a g e o f t h e i n t e r a c t i o n o f t h e unknown i n h i b i t o r , I 2 > w i t h t h e two r a c e m a t e s , I , and S. When, as i n t h e p r e s e n t c a s e , K, = K s ^ 5 , a and y a r e p r o b a b l y s i m i l a r i n magnitude anyway, so t h a t A s h o u l d be a good measure o f e i t h e r e f f e c t . P a r a m e t e r A i s r e a d i l y e x t r a c t e d from t h e s l o p e s o f two Dixon p l o t s o f (1/v) v e r s u s [ I 2 ] c o r r e s p o n d i n g t o two v a l u e s f o r [ S ] = [ I , ] . I f t h e s l o p e s o f t h e two l i n e s a r e d e s i g n a t e d as ( S l o p e ) , and (S l o p e ) , , , and t h e i r r a t i o i s d e f i n e d as ^S 1 1 1 R . S1°pei _ terf * ^  * ^  ( 3 9 ) S l o p e 2 K S ( 1 , 1 , 1 , V m a x K 2 ( ^ 7 « K 1 Y V th e n p a r a m e t e r A i s r e a d i l y o b t a i n e d f r o m 1 A (R - D [ ( l/K,)+(1/K s)] / L _ - _R_ \ (3.10) \CS], CS]2 J ' - 70 -S i n c e K $ = 9.39 x 1 0 " 4 M f o r . L-BAPA, (3.11) and Kj = 8 - 0 0 x 1 0 " 4 M f o r D-BAPA, 1 5 t h e p a r a m e t e r , A, i s e a s i l y o b t a i n e d f r o m t h e Dixon p l o t s shown i n t h e F i g u r e s f o r t h e R e s u l t s and D i s c u s s i o n . As a u s e f u l g u i d e t o t h e p r e s e n t a t i o n , t h e s i g n i f i c a n c e o f D i x o n - i n t e r s e c t i o n p o i n t s and A - v a l u e s i s l i s t e d i n T a b l e 3.1 f o r r e f e r e n c e . D. R e s u l t s and D i s c u s s i o n V a l u e s f o r t h e i n h i b i t i o n c o n s t a n t , Kg, and i n t e r a c t i o n c o e f f i c i e n t , A ( s e e T h e o r y ) f o r e a c h o f t h e seven i n h i b i t o r s o f th e p r e s e n t s t u d y a r e l i s t e d i n T a b l e 3.2. V a l u e s f o r K 2 and A were d e t e r m i n e d from t h e D i x o n p l o t s shown i n F i g u r e 3.1. The s e l f - c o n s i s t e n c y o f t h e d e t e r m i n a t i o n o f A i s e v i d e n c e d by t h e f a c t t h a t a l t h o u g h A was d e t e r m i n e d f r o m t h e r a t i o o f t h e s l o p e s o f t h e two D i x o n p l o t s f o r a g i v e n i n h i b i t o r , t h e v a l u e o f A c o r r e s p o n d s e x a c t l y t o t h e c o r r e c t ( l / v ) - v a l u e a t t h e i n t e r s e c t i o n o f t h e p l o t s ( i . e . , above o r below t h e x - a x i s ) f o r each i n h i b i t o r , as p r e d i c t e d i n t h e T h e o r y s e c t i o n and summarized i n T a b l e 3.1. The d a t a i n T a b l e 3.2 show t h a t o n l y two o f t h e seven compounds t e s t e d a r e t r u e c o m p e t i t i v e i n h i b i t o r s o f t r y p s i n ; t h e r e m a i n i n g compounds a c t u a l l y a c t i v a t e t h e b i n d i n g o f s u b s t r a t e . - 71 -F i g u r e 3.1 Dixon p l o t s f o r i n h i b i t i o n o f t r y p s i n by t h e i n h i b i t o r shown i n each p l o t . O r d i n a t e g i v e s i n i t i a l r e a c t i o n r a t e i n l i t e r m o l e " 1 s e c x 10^; a b s c i s s a i s m o l a r c o n c e n t r a t i o n o f the a p p r o p r i a t e i n h i b i t o r . C o n c e n t r a t i o n o f ( r a c e m i c ) s u b s t r a t e -4 D , L - b e n z o y l a r g i n i n e - p - n i t r o a n i l i d e was: 0.833 x 10 M ( u p p e r l i n e f o r each p l o t ) ; 1.67 x 10~ 4M ( l o w e r l i n e f o r each p l o t ) ; and 2.5 x 10~ 4M ( l o w e s t c u r v e f o r m - m e t h o x y p h e n y l g u a n i d i n e ) . T r y p s i n c o n c e n t r a t i o n was 5.5 x 1 0 ~ 7 M , i n 0.05M t r i s b u f f e r , pH 7.65, t e m p e r a t u r e 3 0 . 0 ° C , a t Ca c o n c e n t r a t i o n o f 0.02 M. - 72 -u 3 / 2 1 I i i i I L_ 20 -10 0| 5 10 15 20 25 30 3-METHOXYPROPYLAMI NE,103M m-METHOX YPHEN YLGU AN IDI NEHCL,104M P-METHOXYPHENYLGUANIDINEHCL.10 M m-ANISIDINEHCL,103M P-ANISIDINEHCL,10JM -10 -5 ACETAMIDI NE, 103M P-METHOXYBENZYLAMI NE,106M - 73 -T a b l e 3.1 I n t e r p r e t a t i o n o f t h e s i g n i f i c a n c e o f t h e y - v a l u e a t t h e i n t e r s e c t i o n o f two D i x o n p l o t s c o r r e s p o n d i n g t o two d i f f e r e n t c o n c e n t r a t i o n s o f a r a c e m i c " s u b s t r a t e " . The " s u b s t r a t e " i t s e l f c o n s i s t s o f one i s o m e r w hich i s a t r u e s u b s t r a t e and t h e o t h e r i s o m e r i s a c o m p e t i t i v e i n h i b i t o r , ( s e e T h e o r y ) . The A - v a l u e d e f i n e d by eq. ( 3 , 8 ) , w i t h e x p e r i m e n t a l d e t e r m i n a t i o n g i v e n as e q . (3 ,10), i n d i c a t e s t h e d e g r e e o f c o o p e r a t i v i t y between b i n d i n g o f an unknown i n h i b i t o r , I 2 > w i t h t h e r a c e m i c " s u b s t r a t e " , S and I , . V a l u e o f (1/v) a t A - v a l u e i n t e r - M e c h a n i s t i c D e s c r i p t i o n s e c t i o n o f two D i x o n p l o t s _ A = °° p o s i t i v e I 2 i s p u r e l y c o m p e t i t i v e w i t h I , and S. 1 < A < °° p o s i t i v e I 2 r e s t r i c t s , b u t does n o t p r e v e n t b i n d i n g o f I, and S. A = 1 z e r o I 2 has no e f f e c t on b i n d i n g o f I , and S. A < 1 n e g a t i v e I 2 f a c i l i t a t e s b i n d i n g o f I, and S. T a b l e 3.2 S t r u c t u r e s , i n h i b i t i o n c o n s t a n t s , and i n t e r a c t i o n c o e f f i c i e n t s f o r b i n d i n g o f i n h i b i t o r s t o t r y p s i r F o r i n t e r p r e t a t i o n o f parameter A, see T a b l e 3 j . E x p e r i ments were c a r r i e d o u t a t pH 7.65 a t 30°C, i n t h e p r e s e n c e o f 0.02M CaCl2 and 0.05M t r i s b u f f e r . Name S t r u c t u r e ' ' K 2 (M) A ac e t a m i d i n e • ^ C — C H 3 3.5 x 10" 0.75 H2N 3-methoxypropylamine + H3NCH2CH2CH2OCH3 2.2 x 10 « H H p-methoxybenzylamine + H 3 N H 2 C _ , \ ^ ^ ~ 0 C H 3 6.1 x 10 4 - » H H p - m e t h o x y p h e n y l g u a n i d i n e H2N^ N - ^ ^ - O C H 3 2.3 x 1 0 " 3 0.5 + f H H H 0CH3 m-methoxyphenylguanidine ^ N - ^ ^ - H 5.0 X 1 0 ~ 4 0.3 H2M* H H H H w p - a n i s i d i n e ^n-^-oc^ 1.7 x l O " 2 H H m - a n i s i d i n e W 3 9.4 x 1 0 ~ 2 0.1 0.17 - 75 -P u b l i s h e d D i x o n p l o t s f o r f i v e a d d i t i o n a l compounds have a l s o been s u b j e c t e d t o t h e same a n a l y s i s , w i t h t h e r e s u l t s g i v e n i n T a b l e F a i l u r e t o i n c l u d e t h e s p e c i e s , EI -j and EI ^  S i n t h e k i n e t i c scheme l e d t h o s e a u t h o r s t o c o n c l u d e t h a t t h e c y c l o h e x y l compounds were c o m p e t i t i v e i n h i b i t o r s when, as t h e p r e s e n t a n a l y s i s c o n c l u s i v e shows, t h o s e two i n h i b i t o r s a c t t o r e s t r i c t , b u t n o t p r e v e n t a c c e s s o f s u b s t r a t e t o i t s b i n d i n g s i t e . I t i s t o be emphasized t h a t t h e p r e s e n t a n a l y s i s r e q u i r e s no a d d i t i o n a l e x p e r i m e n t s beyond t h e o r d i n a r y D i x o n p l o t a t two d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s , b u t o f f e r s t h e a d d i t i o n a l p a r a m e t e r , A, which p r o v i d e s a measure o f t h e c o o p e r a t i v i t y between b i n d i n g o f i n h i b i t o r and b i n d i n g o f t h e r a c e m i c s u b s t r a t e I-j and S. A t t e n t i o n w i l l now be t u r n e d t o t h e s p e c i f i c c o n c l u s i o n s about t r y p s i n w h i c h may be g a t h e r e d f r o m t h e r e s u l t s i n T a b l e s 3.2 and 3.3. F i r s t o f a l l , i t i s c l e a r t h a t a t l e a s t one s e c o n d a r y b i n d i n g s i t e e x i s t s , s i n c e seven i n h i b i t o r s i n t h e T a b l e s can b i n d t o t h e enzyme w i t h o u t p r e v e n t i n g b i n d i n g o f s u b s t r a t e . Comparison o f t h e b i n d i n g o f compounds 4 and 5 o f T a b l e 3.2 shows t h a t t h e c h a r g e d m e t a - d e r i v a t i v e shows t h e s t r o n g e r b i n d i n g , i n c o n t r a s t t o t h e b e h a v i o r f o r c h y m o t r y p s i n , where NMR r e s u l t s have shown t h a t s i m i l a r p a r a - d e r i v a t i v e s a r e more r i g i d l y h e l d 1 c t h a n t h e c o r r e s p o n d i n g m e t a - i n h i b i t o r s . A l t h o u g h t h e r e i s r e a s o n t o e x p e c t t h a t a n e u t r a l i n h i b i t o r o f t r y p s i n m i g h t b i n d a t a s e c o n d a r y s i t e 7 , 1 7 , as w i t h compounds 6 and 7 o f T a b l e 3.2, i t T a b l e 3.3 S t r u c t u r e s , i n h i b i t i o n c o n s t a n t s , and i n t e r a c t i o n c o e f f i c i e n t s f o r b i n d i n g o f i n h i b i t o r s t o t r y p s i n . F o r i n t e r p r e t a t i o n o f p a r a m e t e r A, see Table3.1. A - v a l u e s were c a l c u l a t e d from p u b l i s h e d D i x o n p l o t s ( M a r e s - G u i a and Shaw, 19 6 5 ) , f o r e x p e r i m e n t s c a r r i e d out a t pH 8.15 a t 15°C i n the absence o f C a + + . ompound Name S t r u c t u r e Kg (M) A H»N / = K c 1 p-aminobenzamidine 4 c~\ / - N H « • 8 , 2 5 x 1 0 H,N Nf - / V c 2 phenyl g u a n i d i n e . + y 7.25 x 10 3 HiN HjN • r ' 3 b e n z a m i d i n e . + c - / \ 1.84 x 1 0 " 3 - S c y c l o h e x y l c a r b o x a m i d i ne 4.27 x 10" 18 c y c l o h e x y l g u a n i d i ne H.N^ N- 1.54 x 10 -3 18 - 77 -i s somewhat s u r p r i s i n g t h a t a number o f o t h e r c h a r g e d i n h i b i t o r s s h o u l d be a t t r a c t e d t o s e c o n d a r y s i t e s . I t i s now acknowledged t h a t even h i g h l y p u r i f i e d t r y p s i n e x h i b i t s some c h y m o t r y p s i n - 1 i k e a c t i v i t y toward a r o m a t i c e s t e r 18 19 20 21 h y d r o l y s i s ' ' ' , i n d i c a t i n g the p r e s e n c e o f a b i n d i n g s i t e f o r a r o m a t i c r e s i d u e s c l o s e t o t h e c a t a l y t i c s i t e . In t h i s c o n t e x t , i t i s i n t e r e s t i n g t o n o t e t h a t a l l b u t one o f t h e a r o m a t i c compounds i n t h i s s t u d y showed b i n d i n g a t a s e c o n d a r y s i t e , and t h e b i n d i n g was i n e a c h c a s e c a p a b l e o f a f f e c t i n g t h e b i n d i n g o f s u b s t r a t e . The D i x o n p l o t b e h a v i o r f o r m - m e t h o x y p h e n y l g u a n i d i n e o f f e r s a d d i t i o n a l evidcr.ee c f s e c o n d a r y b i n d i n g ( F i g u r e 3 . 1 ) . When a t h i r d a s s a y was c o n d u c t e d a t a c o n c e n t r a t i o n o f r a c e m i c s u b s t r a t e , -4 D,L-BAPA o f 2.5 x 10 M, i t was o b s e r v e d t h a t t h e D i x o n p l o t c u r v e d downward a t h i g h c o n c e n t r a t i o n o f m - m e t h o x y p h e n y l g u a n i d i n e . The e x p e r i m e n t was r e p e a t e d s e v e r a l t i m e s and t h e e f f e c t was r e p r o d u c i b l e . The downward d e f l e c t i o n c o u l d be due t o enhanced b i n d i n g o f s u b s t r a t e o r t o an a c t i v a t i o n o f t h e h y d r o l y s i s s t e p i t s e l f , and must a r i s e f r o m t h e p r e s e n c e o f an a p p r e c i a b l e amount o f s p e c i e s , E l g S . A f i n a l a s p e c t o f t h e p r e s e n t r e s u l t s i s t h e a p p a r e n t e f f e c t o f C a + + i o n on i n h i b i t o r b i n d i n g . Most r e p o r t e d t r y p s i n i n h i b i t o r s . . .' . . . , .... 8,22,23,24,25,26,27,28,29,30 t o d a t e have a p p e a r e d t o be p u r e l y c o m p e t i t i v e » » » > » » » » » , b u t most s t u d i e s o f t r y p s i n i n h i b i t i o n have been c o n d u c t e d i n t h e 4 - 78 -absence o f Ca . In the p r e s e n t s t u d y , our v a l u e f o r t h e i n h i b i t i o n b i n d i n g c o n s t a n t , Kg, f o r t h e s m a l l m o l e c u l e , a c e t a m i d i n e ( s e e T a b l e 3.2) i s n e a r l y t h e same as o b t a i n e d i n t h e abs e n c e o f c a l c i u m i o n . In c o n t r a s t , b i n d i n g o f t h e a r o m a t i c compounds 3-7 i n T a b l e 3.2 i s much weaker t h a n t h e b i n d i n g o f a n a l o g o u s i n h i b i t o r s i n t h e ++ ++ absence o f Ca found i n T a b l e 3.3. Now Ca has been s u g g e s t e d t o b i n d t o t r y p s i n so as t o h o l d t o g e t h e r two " l o o p s " i n t h e 31 ++ enzyme b i n d i n g o f Ca does i n d u c e a c o n f o r m a t i o n a l change 32 r e s u l t i n g i n a more compact s t r u c t u r e w i t h an accompanying 33 enhancement i n t h e r a t e o f h y d r o l y s i s o f s u b s t r a t e . The b e h a v i o r o f a r o m a t i c i n h i b i t o r s i n T a b l e 3.2 s u g g e s t s t h a t t h e c o n f o r m a t i o n a l change i n d u c e d by C a + + b o t h r e s t r i c t s a c c e s s o f t h e s e i n h i b i t o r s t o t h e s u b s t r a t e - b i n d i n g p o c k e t ( o n l y compound 3 c o u l d g a i n a c c e s s ) , and a l s o f a v o r s b i n d i n g a t s e c o n d a r y s i t e s . A s i m i l a r e f f e c t may a c c o u n t f o r t h e s t a b i l i z a t i o n o f t r y p s i n a g a i n s t a u t o l y s i s i n t h e p r e s e n c e o f C a + + , by making t h e s u b s t r a t e -b i n d i n g s i t e more s e l e c t i v e . E. SUMMARY When a r a c e m i c " s u b s t r a t e " i s us e d i n a s s a y o f an unknown i i n h i b i t o r o f an enzyme, i t may w e l l be t h a t o n l y one o p t i c a l i s o m e r o f t h e " s u b s t r a t e " i s a t r u e s u b s t r a t e , and the o t h e r a c o m p e t i t i v e i n h i b i t o r , as i n t h e p r e s e n t example. The s i t u a t i o n may be e x p l o i t e d as f o l l o w s . D e t a i l e d k i n e t i c a n a l y s i s shows t h a t i n i t i a l v e l o c i t y - 79 -measurements may be used t o c o n s t r u c t a Dixon p l o t i n t h e u s u a l way, and t h e i n t e r s e c t i o n o f two p l o t s c o r r e s p o n d i n g t o two ( r a c e m i c ) " s u b s t r a t e " c o n c e n t r a t i o n s w i l l s t i l l y i e l d t h e i n h i b i t i o n c o n s t a n t f o r the unknown i n h i b i t o r . In a d d i t i o n , however, t h e r a t i o o f t h e s l o p e s o f t h e two p l o t s p r o v i d e s a measure o f t h e i n t e r a c t i o n ( c o m p e t i t i v e , r e p u l s i v e , n o n - c o m p e t i t i v e , o r a t t r a c t i v e ) o f t h e unknown i n h i b i t o r toward t h e " s u b s t r a t e " . The i m p o r t a n c e i s t h a t an i n h i b i t o r may show a p o s i t i v e ( 1 / v ) -v a l u e a t t h e i n t e r s e c t i o n p o i n t o f t h e Dixon p l o t s , when b i n d i n g o f t h a t i n h i b i t o r s t i l l a l l o w s a c c e s s o f s u b s t r a t e t o i t s b i n d i n g s i t e , i n c o n t r a s t t o t h e u s u a l i n t e r p r e t a t i o n o f a D i x o n p l o t i n t e r s e c t i o n . T h i s p o i n t has been d e m o n s t r a t e d u s i n g i n h i b i t o r s modeled a f t e r a r g i n i n e and l y s i n e , and i n a d d i t i o n , t h e method has been used t o p r o v i d e d i r e c t e v i d e n c e f o r s e c o n d a r y b i n d i n g s i t e s on t r y p s i n . The p r e s e n c e o f C a + + a p p e a r s t o f a v o u r b i n d i n g o f a r o m a t i c i n h i b i t o r s t o t h e s e s e c o n d a r y s i t e s , and s e v e r a l i n h i b i t o r s a c t u a l l y a c t i v a t e t h e b i n d i n g o f " s u b s t r a t e " . - 80 -R e f e r e n c e s 1. A. T u l i n s k y , p r i v a t e c o m u n i c a t i o n . 2. D.E. K o s h l a n d , and K.E. Neet, Ann. Rev. Biochem. 37_, 359 (1968) 3. J . L . Webb, Enzyme and M e t a b o l i c I n h i b i t o r s , V o l . 1, Academic P r e s s , N.Y., p. 488 ( 1 9 6 3 ) . 4. B. K e i l , i n The Enzymes, V o l . 3, ed. P.D. B o y e r , Academic P r e s s , N.Y., p. 249 ( 1 9 7 1 ) . 5. K.A. Walsh, Methods i n Enzymology 1_9, 41 ( 1 9 7 0 ) . 6. C.G. T r o w b r i d g e , A. K r e h b i e l , and M. L a s k o w s k i , J r . , B i o c h e m i s t r y 2_, 843 ( 1 9 6 3 ) . 7. B.M. Sanborn, and G.E. H e i n , B i o c h i m . B i o p h y s . A c t a 139, 534 ( 1 9 6 7 ) . 8. M. M a r e s - G u i a , and E. Shaw, J . B i o l . Chem. 24fJ, 1579 ( 1 9 6 5 ) . 9. L. G o r i n i , B i o c h i m . B i o p h y s . A c t a 7_, 318 ( 1 9 5 1 ) . 10. M. D e l a a g e , and M. L a z d u n s k i , B i o c h i m . B i o p h y s . A c t a 105, 523 ( 1 9 6 5 ) . 11. R.A.B. Bannard, A.A. C a s s e l m a n , W.F. C o c k b u r n , and G.M. Brown, Can. J . Chem. 36, 1541 ( 1 9 5 8 ) . 12. E. Heftmann, Chromatography, 2nd e d . , R e i n h o l d P u b l i s h i n g C o r p . p. 56, p. 347 ( 1 9 6 4 ) . 13. S. S a k a g u c h i , J . Biochem. Tokyo 5_, 13, 25 ( 1 9 2 5 ) . 14. C . J . Weber, J . B i o l . Chem. 78, 465 ( 1 9 4 8 ) . 15. B.F. E r l a n g e r , N. Kokowsky, and W. Cohen, A r c h . Biochem. B i o p h y s . 95, 271 ( 1 9 6 1 ) . - 81 -16. J.T. G e r i g , and E.W. B i t t n e r , J . Amer. Chem. Soc. 92_, 5001 ( 1 9 7 0 ) . 17. B.M. Sanborn, and G.E. H e i n , B i o c h e m i s t r y 7_, 3616 (196 8 ) . 18. H.A. R a v i n , P. B e r n s t e i n , and A.M. S e l i g m a n , J . B i o l . Chem. 208, 1 (19 5 4 ) . 19. T. Inagami, and J.M. S t u r t e v a n t , J . B i o l . Chem. 235, 1019 (1960 ) . 20. V. K o s t k a , and F.H. C a r p e n t e r , J . B i o l . Chem. 239_, 1799 ( 1 9 6 4 ) . 21. S. Maroux, M. Rovery, and P. D e s n u e l l e , B i o c h i m . B i o p h y s . A c t a 122, 147 ( 1 9 6 6 ) . 22. B.M. Sanborn, and W.P. B r y a n , B i o c h e m i s t r y 7_, 3624 ( 1 9 6 8 ) . 23. J . J . B e c h e t , M.C. G a r d i e n n e t , and J . Yon, B i o c h i m . B i o p h y s . A c t a 122, 101 ( 1 9 6 6 ) . 24. T. Inagami, and S.S. York, B i o c h e m i s t r y 7., 4045 ( 1 9 6 8 ) . 25. T. Inagami, J . B i o l . Chem. 239, 787 ( 1 9 6 4 ) . 26. A.N. G l a z e r , J . B i o l . Chem. 242_, 3326 ( 1 9 6 7 ) . 27. S.A. B e r n h a r d , and H. G u t f r e u n d , P r o c . N a t l . A cad. U.S.A. 53_, 1238 ( 1 9 6 5 ) . 28. K. Tan i z a w a , S. I s h i , and Y. Kanaoka, Biochem. B i o p h y s . Res. Commun. 32_, 893 ( 1 9 6 8 ) . 29. A. d ' A b l i s , and J . J . B e c h e t , B i o c h i m . B i o p h y s . A c t a 140, 435 (1967 ) . 30. M. Gulzow, H. Mix, and A . J . T r e t t i n , Z. P h y s i o l . Chem. 348, 285 ( 1 9 6 7 ) . 31. R.M. S t r o u d , L.M. Kay,and R.E. D i c k e r s o n , C o l d S p r i n g Harbor Symp. Quant. B i o l . 36_, 125. 32. M. L a z d u n s k i , and M. D e l a a g e , B i o c h i m . B i o p h y s . A c t a 105, 541 ( 1 9 6 5 ) . 33. N.M. Green and H. N e u r a t h , J . B i o l . Chem. 204,379(1953). - 82 -CHAPTER IV The S t u d i e s o f R i g i d i t y o f B i n d i n g o f I n h i b i t o r s t o T r y p s i n by NMR And i t s C o r r e l a t i o n W i t h t h e S t r e n g t h o f B i n d i n g A. I n t r o d u c t i o n As d e t a i l e d i n a number o f r e c e n t r e v i e w s 1 - 6 , t h e r e i s c o n s i d e r a b l e i n t e r e s t and d i s a g r e e m e n t a b o u t t h e r e l a t i v e i m p o r t a n c e o f v a r i o u s e f f e c t s which c o n t r i b u t e t o t h e e f f i c i e n c y and s p e c i f i c i t y o f enzyme c a t a l y s i s . W h i l e c h e m i c a l p r e c e d e n t s f r o m p h y s i c a l o r g a n i c s t u d i e s have p r o v i d e d i n s i g h t toward s u c h a s p e c t s as 7 8 9 " p r o x i m i t y " e f f e c t , a c i d - b a s e c a t a l y s i s , and s o l v e n t e f f e c t s , t h e r o l e o f s e l e c t i v e o r i e n t a t i o n o f s u b s t r a t e i n enhancement o f c a t a l y s i s i s more d i f f i c u l t t o e v a l u a t e . P r e s e n t d i s c u s s i o n o f enzyme f u n c t i o n c e n t e r s around t h e r e l a t i v e i m p o r t a n c e o f s t r a i n , w hether g e o m e t r i c 1 0 o r e l e c t r o n i c 1 1 , 1 2 , v e r s u s o r i e n t a t i o n o r a l i g n m e n t o f the r e a c t a n t s by t h e e n z y m e - - i f t h e o r i e n t a t i o n r e q u i r e m e n t o f a r e a c t i o n i s s u f f i c i e n t l y narrow, t h e n i t i s p o s s i b l e t o a c c o u n t f o r t h e e n t i r e r a t e enhancement w i t h o u t 13 i n v o k i n g any s t r a i n i n t h e r e a c t a n t m o l e c u l e s . Such emphasis on 14 15 t h e i m p o r t a n c e o f o r i e n t a t i o n has been c r i t i c i z e d on the b a s i s - 83 -o f s h a l l o w minima f o r e n e r g y as a f u n c t i o n o f c o n f o r m a t i o n from both e x p e r i m e n t and t h e o r y f o r t r a n s i t i o n s t a t e bonds, but no d i r e c t e v i d e n c e i s a v a i l a b l e . I t i s becoming i n c r e a s i n g l y c l e a r t h a t a m a j o r d e g r e e o f s t r a i n r e s u l t s on b i n d i n g o f s u b s t r a t e s t o most enzymes. X - r a y 1 g d i f f r a c t i o n o f l ysozyme i n t h e p r e s e n c e o f s u b s t r a t e a n a l o g s s u g g e s t e d t h a t r i n g "D" m i g h t be d i s t o r t e d from a c h a i r t o a h a l f - c h a i r c o n f o r m a t i o n on b i n d i n g o f t h e hexamer, ( N - a c e t y l g l u c o s a m i n e : N - a c e t y l m u r a m i c a c i d ) ^ , t o t h e enzyme. N u c l e a r m a g n e t i c r e s o n a n c e (NMR) s t o p p e d - f l o w measurement o f t h e s c a l a r c o u p l i n g c o n s t a n t f o r t h e "D" r i n g o f the a n a l o g o u s t e t r a m e r i c s u b s t r a t e ^ has r e c e n t l y shown d e f i n t i v e l y t h a t t h e r e i s a d i s t o r t i o n o f t h e " D " - r i n g t o a h a l f - c h a i r on b i n d i n g t o lysozyme i n s o l u t i o n . A l o n g s i m i l a r l i n e s , i t i s o f t e n t h e c a s e t h a t an enzyme may change i t s own c o n f o r m a t i o n on b i n d i n g o f s u b s t r a t e , as i l l u s t r a t e d 1 o by X - r a y c r y s t a l l o g r a p h y o f c a r b o x y p e p t i d a s e A , and as d e m o n s t r a t e d 19 i n s o l u t i o n by p r o t o n NMR r e l a x a t i o n work w i t h c r e a t i n e k i n a s e . R e g a r d i n g t h e o r i e n t a t i o n q u e s t i o n , i t has l o n g been r e c o g n i z e d t h a t m a g n e t i c r e s o n a n c e r e l a x a t i o n t i m e s can p r o v i d e d i r e c t i n f o r m a t i o n a b o u t m o t i o n a l l a b i l i t y f o r a m o l e c u l e ; however, t h e s m a l l changes o f 1^ °f t n e s h a r p r e s o n a n c e l i n e ( s u c h as methyl group ) upon a d d i n g enzyme i s d i f f i c u l t t o e s t i m a t e by c o n v e n t i o n a l l i n e w i d t h measurement, s i n c e t h e c o n t r i b u t i o n t o the l i n e w i d t h f r o m m a g n e t i c f i e l d i n h o m o g e n e i t y can be s e v e r a l - 84 -t i m e s l a r g e r t h a n t h a t f r o m e i t h e r t h e m o l e c u l a r t r a n s v e r s e r e l a x a t i o n . t i m e o r t h e change i n d u c e d by t h e enzyme. 20 21 O n l y r e c e n t l y , however, have e x p e r i m e n t a l and t h e o r e t i c a l methods become a v a i l a b l e f o r measurement and i n t e r p r e t a t i o n o f r e l a x a t i o n t i m e s f o r i n d i v i d u a l h i g h - r e s o l u t i o n p r o t o n NMR s i g n a l s i n l i q u i d s . In t h i s s t u d y , a s e r i e s o f t r y p s i n i n h i b i t o r s has been c o l l e c t e d and/or s y n t h e s i z e d ( d e s c r i b e d i n C h a p t e r I I I ) , such t h a t e a c h i n h i b i t o r p o s s e s s e s a s i n g l e , s h a r p ( u n s p l i t ) p r o t o n NMR s i g n a l f r o m a methyl group t o g i v e unambiguous i n t e r p r e t a t i o n - - N M R r e l a x a t i o n f o r i n h i b i t o r i n t h e p r e s e n c e o f enzyme t h e n p r o v i d e s a measure o f t h e r i g i d i t y w i t h w h i c h t h a t i n h i b i t o r i s bound t o t h e enzyme. To h e l p i d e n t i f y t h e b i n d i n g s i t e , complementary s t e a d y - s t a t e k i n e t i c s s t u d i e s were c o n d u c t e d — b y u s i n g a r a c e m i c s u b s t r a t e ( s e e C h a p t e r I I I ) . F i n a l l y , by c o r r e l a t i n g r i g i d i t y o f b i n d i n g w i t h s t r e n g t h o f b i n d i n g , and b i n d i n g s t r e n g t h w i t h c a t a l y t i c e f f i c i e n c y , one can compare r i g i d i t y o f b i n d i n g w i t h c a t a l y t i c enhancement t o c l a r i f y t h e r o l e o f r i g i d o r i e n t a t i o n i n enzyme a c t i o n . B. E x p e r i m e n t a l  1. I n s t r u m e n t P u l s e d H i g h R e s o l u t i o n NMR S p e c t r o m e t e r A v a r i a n HA-100 NMR s p e c t r o m e t e r had been m o d i f i e d t o p e r f o r m t h i s s e l e c t i v e d e t e r m i n a t i o n o f r e l a x a t i o n t i m e - 85 -a c c o r d i n g t o Freeman's p r o c e d u r e * ^ . The l o c k c h a n n e l f r e q u e n c y i s v a r i a b l e and i s t a k e n f r o m a d i g i t a l f r e q u e n c y s y n t h e s i z e r (model 51106) d r i v e n by a 1 MHz m a s t e r o s c i l l a t o r . The l o c k s i g n a l o f ca_. 2.5 KHz was o b t a i n e d by d i g i t a l d i v i s i o n o f a r a d i o f r e q u e n c y i n t h e MHz r a n g e and f i l t e r i t o o b t a i n a s i n e wave a t (1 v.p.p.) whose f r e q u e n c y can be s e t w i t h a c c u r a c y and w h i c h can be s w i t c h e d t o r e p l a c e t h e o r d i n a r y manual o s c i l l a t o r ( c a r d 910868). In t h e o b s e r v a t i o n c h a n n e l , t h e 2.5 KHz a u d i o - m o d u l a t i o n f r e q u e n c y r e q u i r e d f o r d e t e c t i o n o f r e s o n a n c e s was d e r i v e d by d i g i t a l d i v i s i o n o f t h e 1 MHz m a s t e r o s c i l l a t o r and i s f i x e d . Hence t h e l o c k - i n a m p l i f i e r ( P r i n c e t o n a p p l i e d r e s e a r c h - 1 2 1 ) was p e r m a n e n t l y tuned t o t h i s f r e q u e n c y . P u l s e I and p u l s e I I a r e t h e e x a c t same f r e q u e n c y 2.5 KHz, e x c e p t p u l s e I I can have a phase s h i f t ( 9 0 ° , 180°) r e l a t i v e t o p u l s e I i n t r o d u c e d m a n u a l l y . The p u l s e s I and II a r e g a t e d a c c o r d i n g t o s w i t c h s e t t i n g s on the f r o n t panel t o g i v e any sequence d e s i r e d . T h e s e p u l s e sequences a r e a m p l i f i e d t h r o u g h a H e w l e t t - P a c k a r d 465 A a m p l i f i e r , and t h e y f e d t o t h e D.C. M o d u l a t i o n C o i l s i n t o t h e p r o b e . The demodulated o u t p u t o f t h e r e c e i v e r c o i l ( d e m odulated i n V-4511 r e c e i v e r u n i t ) i s f e d t o t h e l o c k i n a m p l i f i e r (PAR 121). The s i g n a l was r e c o r d e d on a T e k t r o n i x (R 564 B) s t o r a g e scope and p h o t o g r a p h e d by a P o l a r o i d Camera. The p r o c e d u r e and t h e l i m i t a t i o n s o f t h e s e methods have been - 86 -t h o r o u g h l y d e s c r i b e d e l s e w h e r e " ^ 1 , Here o n l y some a d d i t i o n a l e x p e r i e n c e s o f my own w i l l be r e p o r t e d . (a) In s p i t e o f r e f o c u s i n g n a t u r e o f t h e s p i n echo p u l s e t o s e q u e n c e s , i t i s s t i l l n e c e s s a r y , p r i o r t h e p u l s i n g e x p e r i m e n t , t o a d j u s t t h e f i e l d h o m o g e n e i t y , p h a s i n g , and o t h e r c o n t r o l s such t h a t a s y m m e t r i c a l s i g n a l i s o b t a i n e d on-sweeping t h r o u g h r e s o n a n c e f r o m e i t h e r u p - or d o w n - f i e l d i n c o n v e n t i o n a l c o n t i n u o u s -wave o p e r a t i o n . (b) The c o n c e n t r a t i o n o f t h e i n t e r n a l s t a n d a r d ( f o r l o c k i n g t h e f i e l d f r e q u e n c y r a t i o ) s h o u l d n o t be t o o h i g h . O t h e r w i s e , t h e d u r i n g t h e p u l s e , A r e c e i v e r w i l l be s a t u r a t e d and l o s e t h e " l o c k " . ( c ) No NMR l i n e s h o u l d be c l o s e r t h a n 30 Hz t o t h e p u l s e d l i n e ; a l s o , the f a r t h e r t h e l o c k s i g n a l f r o m t h e o b s e r v e d l i n e , t h e b e t t e r t h e r e s u l t s . (d) A S/N o f a t l e a s t 3:1 was r e q u i r e d t o r e c o r d a r e l i a b l e C a r r - P u r c e l l sequence s p i n - e c h o t r a i n . (e) F o r t h e c a s e , Tg < 1 s e c , t h e p u l s e power needed t o g e n e r a t e a TT o r TT/2 p u l s e c o u l d s a t u r a t e t h e r e c e i v e r c o i l . The r e s u l t w i l l be a u n s t e a d y " l o c k " and a w a n d e r i n g m a g n e t i c f i e l d , so t h a t t h e t r a c e o f t h e r e l a x a t i o n on t h e o s c i l l o - s c o p e w i l l n o t be r e p r o d u c i b l e . One can t h e n measure t h e d e c a y o f t h e peak o f i n t e r e s t and t h e c o n t r i b u t i o n o f t h e f i e l d i n h o m o g e n u i t y c a n be e s t i m a t e d from t h e f r e e i n d u c t i o n d e c a y o f an i n t e r n a l s t a n d a r d , u s u a l l y - 87 -t e r t i a r y - b u t y l a l c o h o l p r e s e n t i n t h e same sample t u b e . 2. Method o f Measurement a. C h e m i c a l S h i f t F o r t h e measurement o f c h e m i c a l s h i f t s , a c a p i l l a r y c o n t a i n i n g t e t r a m e t h y l s i l a n e as r e f e r e n c e s t a n d a r d was i n s e r t e d i n t o t h e sample t u b e s . The c a p i l l a r y was h e l d c o n c e n t r i c w i t h t h e NMR t u b e and m a g n e t i c f i e l d was " l o c k e d " by u s i n g t h i s e x t e r n a l s t a n d a r d ; c h e m i c a l s h i f t s were measured w i t h r e f e r e n c e t o t h e m e t h y l p r o t o n s o f an i n t e r n a l s t a n d a r d , u s u a l l y t e r t i a r y b u t y l a l c o h o l o r a c e t o n e . The sweep r a t e f o r c h e m i c a l s h i f t measurements was 0.2 Hz/sec o r l e s s , t o s a t i s f y t h e slow sweep c o n d i t i o n n e c e s s a r y f o r a c c u r a t e c h e m i c a l s h i f t d e t e r m i n a t i o n . The f r e q u e n c y o f t h e peak o f i n t e r e s t and a l s o t h a t o f the i n t e r n a l s t a n d a r d was o b t a i n e d by i n t r a p o l a t i o n between two f r e q u e n c y m a r k e r s on b o t h s i d e s o f t h e peak t o be measured. The f r e q u e n c y o f t h e marks was r e a d a c c u r a t e l y f r o m a f r e q u e n c y c o u n t e r ( H e w l e t t - P a c k a r d 3734 A ) . b. R e l a x a t i o n Time T r a n s v e r s e m a g n e t i c r e l a x a t i o n t i m e , ( T g ) , was d e t e r m i n e d f o r t h e methyl p r o t o n NMR s i g n a l f o r e a c h i n h i b i t o r by t h e u s u a l 23 G i l l - M e i b o o m m o d i f i c a t i o n o f a C a r r - P u r c e l l p u l s e sequence Measurements 1 o f l o n g i t u d i n a l r e l a x a t i o n t i m e , T 1 , were p e r f o r m e d by a TT - t - IT/2 p u l s e s e q u e n c e 2 4 , and v i s c o s i t y c o r r e c t i o n s t o - 88 -Tg were shown t o be n e g l i g i b l e . Each r e p o r t e d T, o r Tg r e p r e s e n t s an a v e r a g e o f a t l e a s t 6 i n d e p e n d e n t d e t e r m i n a t i o n s . The t e m p e r a t u r e o f t h e probe was d e t e r m i n e d d i r e c t l y by h a v i n g a t h e r m i s t o r (YSI model 4256 T e l e - T h e r m o m e t e r ) i n t h e NMR t u b e t h e r m a l l y e q u i l i b r a t e d w i t h i n t h e sample " p r o b e " ; i t was 30 + 1°C. In o r d e r t o f u l f i l l t h e 30 H z - s p a c i n g c o n d i t i o n s e t by t h e p u l s e u n i t m e n t i o n e d e a r l y i n t h e i n s t r u m e n t a l s e c t i o n , t h e t y p i c a l 0.05M TRIS b u f f e r was sometimes r e p l a c e d by O.IM b o r a t e b u f f e r t o a v o i d any s t r o n g s i g n a l w i t h i n 30 Hz o f t h e o b s e r v e d r e s o n a n c e . M a g n e t i c f i e l d was s t a b i l i z e d by l o c k i n g o n t o an HDO s i g n a l o r t e r t i a r y b u t y l a l c o h o l s i g n a l d e p e n d i n g on which s i g n a l was f a r t h e r f r o m t h e o b s e r v e d s i g n a l . D e u t e r a t e d methanol was added ( l e s s t h a n 1 5 % ) , whenever i t was needed t o b r i n g enough i n h i b i t o r i n t o t h e s t o c k s o l u t i o n . A l l i n h i b i t o r s o l u t i o n s a r e p r e p a r e d i n t h e f o l l o w i n g way. P r e p a r e a c o n c e n t r a t e d («^0.2M) i n h i b i t o r s t o c k s o l u t i o n as w e l l as c o n c e n t r a t e d b u f f e r s o l u t i o n , C a C l g s o l u t i o n , e t c . The p r o p e r amount o f d i f f e r e n t c o n c e n t r a t e s t o c k s o l u t i o n s was mixed and t i t r a t e d t o pH=7.65 (pD = 8.05 - 8 . 1 ) , t h e t i t r a t e d s o l u t i o n was t h e n b r o u g h t t o t h e volume i n a v o l u m e t r i c f l a s k w i t h DgO. 12 mg TRYPSIN was weighed i n a 5 ml beaker and d i s s o l v e d i n 0.5 ml o f t h e above p r e p a r e d s t o c k s o l u t i o n . The d i s s o l v e d s o l u t i o n was t h e n t r a n s f e r r e d i n t o t h e NMR tube and e q u i l i b r a t e d i n a c o n s t a n t t e m p e r a t u r e b a t h (31°C) f o r 4 m i n u t e s - 89 -and was e q u i l i b r a t e d a g a i n i n t h e s p e c t r o m e t e r f o r a t l e a s t 3 m i n u t e s . The t i m e between d i s s o l v i n g o f t h e enzyme i n the i n h i b i t o r s o l u t i o n and the t i m e f o r -complete r e c o r d i n g t h e d a t a , was l e s s t h a n 25 m i n u t e s . T h i s p r o c e d u r e m i n i m i z e s danger o f a u t o l y s i s o f t r y p s i n (enyzme s t i l l has 95% o f o r i g i n a l a c t i v i t y ) . C. R e s u l t s and D i s c u s s i o n F o r each i n h i b i t o r , p l o t s o f r e c i p r o c a l r e l a x a t i o n t i m e , ( l / T - j ) , ( 1 / T 2 ) , v e r s u s i n i t i a l c o n c e n t r a t i o n r a t i o , ( [ E Q ] / [ I ] 0 ) , o f enzyme t o i n h i b i t o r a r e shown i n F i g u r e 4.2 t o 4.8. F o r some i n h i b i t o r s , t h e p l o t c u r v e s downwards a t h i g h [ E ] / [ I ] r a t i o due t o a r e l a t i v e l y weak b i n d i n g c o n s t a n t . A computer s i m u l a t i o n o f the p r e d i c t e d changes o f l / T - j , l / T ^ a t d i f f e r e n t [E ] / [ I Q ] r a t i o f o r i n h i b i t o r s w i t h v a r i o u s b i n d i n g c o n s t a n t s i s p r e s e n t e d i n F i g u r e 4.1; one can see t h a t t h e " p l o t s " do n o t i n g e n e r a l y i e l d 25 26 a s t r a i g h t l i n e a s one u s u a l l y assumed i n t h e l i t e r a t u r e ' . The p l o t t e d l i n e s f o r a weak i n h i b i t o r ( d i s s o c i a t i o n c o n s t a n t i s l a r g e r t h a n 10 ) w i l l c u r v e downward, whereas the p l o t f o r a s t r o n g i n h i b i t o r i s more o r l e s s a s t r a i g h t l i n e . S i n c e t h e b i n d i n g c o n s t a n t e s t i m a t e d f r o m t h e NMR e x p e r i m e n t i s based on the c u r v a t u r e o f t h e p l o t t e d l i n e s , i t i s e v i d e n t t h a t t h e e s t i m a t i o n o f b i n d i n g c o n s t a n t by NMR w i l l be i n a c c u r a t e f o r t h e s t r o n g i n h i b i t o r s . T h i s i s f u r t h e r e v i d e n c e d when t h e b i n d i n g c o n s t a n t f o r each o f t h e i n h i b i t o r s was e s t i m a t e d by a n o n - l i n e a r l e a s t s q u a r e f i t (BMD - x 85) t o t h e NMR d a t a . - 90 -Figure 4.1 Computer simulation for 1/T2 versus 0 /J-J ] X 103 a t different hypothetical concentration with [E_] •= 10~3 M/A. case A : KI = 10 case B : KI = 10 case C " KI = 10 case D = 10 I_ = J _ + fB r T T '2 '2A '2B With the assumption that the enzyme inhibitor complex is 1:1, J — = 0.5 sec"^, ^J— = 60 sec"^ (rigidly bound *2A '2B inhibitor) fg can be calculated for cases A, B, C, and D, and y— can then be evaluated and plotted against ^ ° " V [ i 0 ] x ]Q3. - 91 -[E] o/[l] ox10 3 - 92 -F i g u r e 4.2 t o F i g u r e 4.8 l / T g and 1/T^ f o r the methyl p r o t o n s o f t h e i n h i b i t o r s a t d i f f e r e n t c o n c e n t r a t i o n s i n t h e p r e s e n c e o f c o n s t a n t amounts o f t r y p s i n , [ E Q ] = 1 0 " 3 • M / i , pD = 8.1. T e m p e r a t u r e 30 ± 1°C. R e l a x a t i o n t i m e s were measured w i t h HA-100 e q u i p p e d w i t h a p u l s e u n i t ( s e e e x p e r i m e n t a l s e c t i o n ) . & 1/T 2  1 / T 1 - 93 -F i g u r e A,. 2 . 6 [E ] o/[l]ox10 3 1 ACETAMIDI NE 2 * + f H 2N — C H 3 - 94 -F i g u r e 4.3 2 3-METHOXYPROPYLAMI NE + H 3 NCH2CH2CH20CH3 - 95 -F i g u r e 4.4 0 10 20 30 40 [E] o / [ l ]oXl0 3 3 P-METHOXYBENZYLAMI NE + H 3 N H 2 C ~ ^ J ^ ~ 0 C H 3 H H H H - 96 -F i g u r e 4.5 \ - 6 [E ]o / [ l ] o *10 p-methoxyphenylguanidine H 2 N H — " U ^ y - OCH H N .C H H F igure 4.6 - 97 -F i g u r e 4.7 F i g u r e 4.8 - 99 -- TOO -T h e o r e t i c a l l y , 1/T, d a t a w i l l y i e l d t h e same i n f o r m a t i o n as l / T g d a t a , f o r d i p o l e - d i p o l e r e l a x a t i o n p r o c e s s e s and t h u s would be a good d o u b l e check f o r the e x p e r i m e n t . But as can be seen i n t h e F i g u r e s 4.2-4.8 t h e changes i n 1/T, i s r a t h e r t o o s m a l l f o r t h i s a p p r o a c h t o be f e a s i b l e . No c h e m i c a l s h i f t was o b s e r v e d f o r any o f t h e i n h i b i t o r s s t u d i e d , t h u s t h e o b s e r v e d r e l a x a t i o n t i m e c a n be d e s c r i b e d as i n c h a p t e r I , c a s e A. (}-) = r - + r - r - r ( 4 J ) '2 *2A 12B T B When t h e t e m p e r a t u r e c o n t r o l e x p e r i m e n t was c o n d u c t e d a t a lower t e m p e r a t u r e ( 1 5 ° C ) , ( l / T g ) o f a l l t h e i n h i b i t o r s become l a r g e r : t h i s shows t h a t t h e f a s t exchange l i m i t a p p l i e s , so t h a t t h e r e l a x a -t i o n t i m e a t t h e bound s i t e d o m i n a t e s t h e t r a n s v e r s e r e l a x a t i o n t i m e , 12 12A 12B In p r i n c i p l e , K, and l / T g g can be e s t i m a t e d by t h e n o n - l i n e a r l e a s t s q u a r e f i t (BMDX-85). But i t was soon f o u n d t h a t t h e d a t a can be f i t w i t h wide range v a l u e s o f Kj f o r a s t r o n g i n h i b i t o r ( K j < 10" ) . Thus t h e bound r e l a x a t i o n t i m e s were c a l c u l a t e d u s i n g the b i n d i n g c o n s t a n t f r o m uv s t u d i e s i n c h a p t e r I I I . For c o m p a r i s o n , some o f the " l e s s p r e c i s e " NMR based K j - v a l u e s a r e a l s o shown i n T a b l e 4.1. - 1 0 1 -To s e t t h e s c a l e f o r t h e ( l / T 2 ) - v a l u e s i n t h e T a b l e 4.1 one can c a l c u l a t e t h e v a l u e w h i c h would be e x p e c t e d i f t h e i n h i b i t o r m o l e c u l e were r i g i d l y bound t o t h e "backbone" o f t r y p s i n , o 07 u s i n g a p r o t o n - p r o t o n d i s t a n c e o f 1.8 A f o r a methyl g r o u p , -8 28 a r o t a t i o n a l c o r r e l a t i o n t i m e o f 1.6 x 10 s e c f o r t r y p s i n , Larmor f r e q u e n c y o f 2TT x 1 0 8 s e c - 1 , and t h e a p p r o p r i a t e f o r m u l a 2 1 1 5 t o g i v e a r i g i d l y bound v a l u e o f ( 1 / T 2 ) = 240 s e c - 1 . More 21 a r e a l i s t i c a l l y , i t i s r e a s o n a b l e t o suppose t h a t even a r i g i d l y h e l d i n h i b i t o r w i l l s t i l l e x h i b i t r a p i d i n t e r n a l r o t a t i o n f o r t h e -CH 3 o r -0CH 3 g r o u p i t s e l f — t h i s w i l l r e s u l t i n a r e d u c t i o n o f t h e a p p a r e n t ( l / T 2 ) - v a l u e by a f a c t o r o f [(3COS 2(IT/2) - l ) / 2 ] 2 = (1/4) t o p r o d u c e an a p p a r e n t "bound" ( l / T g ) o f about 60 s e c - 1 , o r j u s t about what i s o b s e r v e d f o r t h e most r i g i d l y bound i n h i b i t o r s i n T a b l e 4.1. From t h e f a c t t h a t the c o m p e t i t i v e i n h i b i t o r s , 3-methoxy-p r o p y l a m i n e and p - m e t h o x y b e n z y l a m i n e , a r e s t i l l r o t a t i o n a l l y l a b i l e ( T a b l e 4.1) a b o u t t h e l o n g a x i s o f each i n h i b i t o r w i t h i n t h e a c t i v e s i t e , i t can be deduced t h a t any c o n f o r m a t i o n a l change 29 i n d u c e d by t h e f o r m a t i o n o f t h e e n z y m e - i n h i b i t o r complex a l o n g t h e s p e c i f i c b i n d i n g p o c k e t i s n o t so much as t o t o t a l l y i m m o b i l i z e t h e i n h i b i t o r . The s i z e and t h e shape o f t h e c o m p e t i t i v e i n h i b i t o r i s v e r y s i m i l a r t o t h e s p e c i f i c r e s i d u e s , a r g i n i n e o r l y s i n e , f o r w h i c h an e s t e r o r p e p t i d e bond o f a s y n t h e t i c o r n a t u r a l s u b s t r a t e i s h y d r o l i z e d . T h u s , i t a p p e a r s t h a t c o m p l e t e i m m o b i l i z a t i o n o f s u b s t r a t e i s n o t a c r i t i c a l r e q u i r e m e n t f o r t r y p s i n t o c a r r y o u t t h e h y d r o l y s i s . -102-T a b l e 4.1 S t r e n g t h o f b i n d i n g ( K j ) , c o o p e r a t i v i t y toward b i n d i n g o f s u b s t r a t e ( A ) , and r i g i d i t y o f b i n d i n g ( l / T g ) o f v a r i o u s i n h i b i t o r s t o t r y p s i n . Kj = [ E ] [ I ] / [ E I ] . A has t h e f o l l o w i n g i n t e r p r e t a t i o n : A = », i n h i b i t o r i s c o m p e t i t i v e w i t h s u b s t r a t e ; 1 < A < °°, i n h i b i t o r r e s t r i c t s b u t does n o t p r e v e n t b i n d i n g o f s u b s t r a t e ; A = 1, i n h i b i t o r b i n d i n g has no e f f e c t on s u b s t r a t e b i n d i n g ; A < 1, i n h i b i t o r f a c i l i t a t e s b i n d i n g o f s u b s t r a t e . A l a r g e r v a l u e o f ( l / T g ) c o r r e s p o n d s t o more r i g i d b i n d i n g ( s e e t e x t ) : ( 1 / T 2 ) = TTAV, where Av i s t h e n u c l e a r m a g n e t i c r e s o n a n c e l i n e -w i d t h a t h a l f h e i g h t f o r t h e methy p r o t o n s o f t h a t i n h i b i t o r . K T* and ( 1 / T 9 ) * a r e o b t a i n e d from NMR d a t a o n l y ( s e e T e x t ) , ompound Name acetamidine Structure H 2 N 3-methoxypropylamine p-methoxybenzylamine 3.5 x 10~ 'M + H 3 N C H 2 C H 2 C H 2 O C H 3 2 . 2 X 1 0 " 2 H H + H 3 N H 2 C - < ^ ^ - O C H 3 6.1 X 1 0 " 4 H H p-methoxyphenylguanidine H 2 N N - < ^ ^ - O C H 3 + X 2.3 x 10" H N H H H 0CH3 m-methoxyphenylguani di ne H 2 N Y H H 5.0 X 10" p-anisidine m-anisidine H H W H2N-<j3^~0CH3 9«4 X 10" H H H 0CH3 H 2 N - U ;/-H 1.7 X 10" H H A ( 1 / T 2 ) ( 1 / T 2 ) 0.75 <5 sec 12 ± 3 ( 9 . 3 4 . 4 ) 1 8 . 5 ± 4 X l O " 3 36 ± 5 ( 7 . 7 3 .4 ) 44 ± 9 x l O ' 3 0.5 56 ± 7 0 .3 67 ± 6 0 .17 68 ± 22 ( 7 . 7 5 ) 56 ± 20 x l O " 3 0.1 36 ± 4 ( 1 . 6 0.1 ) 55 ± 8 x l O " 2 - 104 -B e f o r e drawing any c o n c l u s i o n s about t h e r i g i d i t y o f b i n d i n g and s t r e n g t h o f b i n d i n g , l e t us r e c o n s i d e r the meaning o f t h e b i n d i n g c o n s t a n t o b t a i n e d f r o m t h e uv s t e a d y - s t a t e s t u d i e s . The b i n d i n g c o n s t a n t from uv s t u d i e s o n l y t e l l s us the s t r e n g t h o f b i n d i n g o f t h e s i t e s w h ich w i l l i n t e r f e r e w i t h t h e h y d r o l y s i s , whereas b i n d i n g w h i c h may have no i n f l u e n c e on t h e h y d r o l y s i s i s not r e f l e c t e d i n t h e uv s t u d i e s . In c o n t r a s t , NMR r e f l e c t s t h e s t a t i s t i c a l l y a v e r a g e d b i n d i n g o f a l l s i t e s no m a t t e r whether t h e bound m o l e c u l e has any f o r m o f i n t e r a c t i o n w i t h t h e s u b s t r a t e a t t h e a c t i v e s i t e o r n o t . O n l y f o r a t r u l y c o m p e t i t i v e i n h i b i t o r (which b i n d s o n l y a t t h e a c t i v e s i t e o f t h e enzyme) w i l l t h e s e two p h y s i c a l t e c h n i q u e s p r o v i d e t h e same b i n d i n g c o n s t a n t . F o r t h e two p u r e l y c o m p e t i t i v e i n h i b i t o r s (compound 2 and 3) 2 3- methoxypropyiamine + H 3 NCH2CH2CH2OCH3 H H 3 p- methoxybenzilamlne H H - 105 -ha v i n g d i f f e r e n t l e n g t h s but s i m i l a r o v e r a l l symmetry, i t i s seen t h a t s t r o n g e r b i n d i n g c o r r e l a t e s d i r e c t l y w i t h g r e a t e r r i g i d i t y a t t h e b i n d i n g s i t e f o r b o t h s e t s o f d a t a (uv and NMR) ( T a b l e 4 . 1 ) . A s i m i l a r c o n c l u s i o n a p p l i e d t o t h e c h a r g e d but n o t p u r e l y c o m p e t i t i v e i n h i b i t o r s (compound 1 and homologs 4 and 5 ) . acetamidine H 2 N •CH, p-methoxyphenylguanidine H H H 2 \ / ^ - ^ H O C H 3 H r f H H H O C H , m-methoxyphenylguanidine H 2 N * H H A r e v e r s e s i t u a t i o n was f o u n d f o r t h e n e u t r a l i n h i b i t o r s (compound 6 and 7) 6 ' p-anisidine H H H H m-anasidine H 0CH 3 H H - 106 -M e t a - a n i s i d i n e , a weaker i n h i b i t o r , i s n e v e r t h e l e s s bound more r i g i d l y t h a n P - a n i s i d i n e , a s t r o n g e r i n h i b i t o r ( T a b l e 4.1). I t was s u g g e s t e d t h a t t h e b i n d i n g s i t e s f o r n e u t r a l compounds i s 29 30 31 d i f f e r e n t f r o m t h e b i n d i n g s i t e s f o r t h e c h a r g e d compounds ' ' . P erhaps the geometry o f n e u t r a l homolog b i n d i n g s i t e s s t o p s t h e * meta-isomer from r o t a t i n g due t o t h e s t e r i c h i n d r a n c e and r e s u l t s t h e r e v e r s e d s i t u a t i o n . A l t h o u g h t h e f a c t o r o f s t e r i c h i n d r a n c e may p l a y a b i g r o l e i n t h e i m m o b i l i z a t i o n o f t h e s m a l l m o l e c u l e a t t h e b i n d i n g s i t e , n e v e r t h e l e s s , t h e g e n e r a l t r e n d r e v e a l e d by t h e NMR r e l a x a t i o n r e s u l t s f o r t h e c h a r g e d i n h i b i t o r i s t h a t t h e s t r o n g e r b i n d i n g c o r r e l a t e s d i r e c t l y w i t h g r e a t e r r i g i d i t y a t t h e b i n d i n g s i t e . S i n c e t h e mechanism o f c a t a l y s i s by t r y p s i n and a-chymotrypsin i s b e l i e v e d t o be t h e s a m e ^ , and f o r c h y m o t r y p s i n , t h e r e l a t i v e m a gnitude o f t h e b i n d i n g c o n s t a n t i s i n d e p e n d e n t o f t h e magni-32 t u d e o f t h e r a t e enhancement . More t o t h e p o i n t , a s m a l l e r 33 34 amount o f d a t a f o r t r y p s i n s u b s t r a t e s ' shows t h a t b i n d i n g c o n s t a n t and c a t a l y t i c e f f i c i e n c y a r e u n r e l a t e d f o r t r y p s i n a l s o . I t w o u ld thus a p p e a r t h a t r i g i d i m m o b i l i z a t i o n may n o t be a major f a c t o r i n enzyme c a t a l y s i s , a t l e a s t f o r t r y p s i n i n t h e s e p r e l i m i n a r y s t u d i e s . * I t s h o u l d be n o t e d t h a t even f o r a s e r i e s o f t r u l y c o m p e t i t i v e i n h i b i t o r s , each o f t h e same b i n d i n g c o n s t a n t s , a d i r e c t c o r r e l a t i o n between t h e s t r e n g t h o f b i n d i n g and t h e r i g i d i t y o f t h e bound s p e c i e s c o u l d be m o d i f i e d by t h e p r e s e n c e o f - s t e r i c h i n d r a n c e . F o r example, a r o d - s h a p e d m o l e c u l e i n a c l e f t c o u l d have a r o t a t i o n a b o u t i t ' s l o n g a x i s , but t h e a d d i t i o n o f an e x t r a s i d e c h a i n g r o u p m i g h t s t o p i t r o t a t i n g c o m p l e t e l y , b u t s t i l l p e r m i t b i n d i n g . - 107 -D. Summary S e v e r a l a r g i n i n e o r l y s i n e a n a l o g i n h i b i t o r s o f t r y p s i n have been s u b j e c t e d t o n u c l e a r m a g n e t i c r e s o n a n c e measurements. The u.v. a s s a y s p r o v i d e d e t e r m i n a t i o n o f t h e b i n d i n g c o n s t a n t o f i n h i b i t o r t o enzyme and i n d i c a t e s t h e t y p e o f b i n d i n g . The NMR r e s u l t s can be a n a l y z e d t o y i e l d a q u a n t i t a t i v e measure o f the d e g r e e o f m o t i o n a l f r e e d o m a t t h e b i n d i n g s i t e o f t h e i n h i b i t o r . S t r e n g t h o f b i n d i n g i s f o u n d t o c o r r e l a t e d i r e c t l y w i t h r i g i d i t y o f b i n d i n g f o r t h e c h a r g e d g r o u p s and s i n c e s t r e n g t h o f b i n d i n g o f s u b s t r a t e does n o t c o r r e l a t e w i t h c a t a l y t i c e f f i c i e n c y , t h e r e s u l t s s u g g e s t t h a t r i g i d i m m o b i l i z a t i o n o f s u b s t r a t e i s n o t a m a j o r f a c t o r i n t r y p s i n c a t a l y s i s . - 108 -R e f e r e n c e s 1. H. G u t f r e u n d and J.R. Knowles, E s s a y s Biochem. 3_> 25 ( 1 9 6 7 ) . 2. W.N. L i p s c o n b , Chem. Soc. Rev. U_, 319 ( 1 9 7 2 ) . 3. D.E. K o s h l a n d , J r . , and K.E. Neet, Ann. Rev. Biochem. 37_, 359 ( 1 9 6 8 ) . 4. R. Lumry, Adv. Chem. Phys. 21_, 567 (1971 ). 5. G.P. Hess and J.A. R u p l e y , Ann. Rev. Biochem. 4TJ, ( 1 9 7 1 ) . 6. D.M. Blow and T.A. S t e i t z , Ann. Rev. Biochem. 39, ( 1 9 7 0 ) . 7. T . B r u i c e and S. B e n k o v i c , i n B i o o r g a n i c Mechanisms, W.A. Be n j a m i n , N.Y. ( 1 9 6 6 ) . 8. M.L. Bender and F . J . Kezdy, Ann. Rev. Biochem. 34, 49 ( 1 9 6 5 ) . 9. J . F . B r a n d t s , i n B i o l o g i c a l M a c r o m o l e c u l e s , e d. S.N. T i m a s h e f f , G.D. Fasman 2, 213 ( 1 9 6 9 ) . 10. W.P. J e n c k s , i n C u r r e n t A s p e c t s o f B i o c h e m i c a l E n e r g e t i c s , ed. N.O. K a p l a n , E.P. Kennedy, Academic P r e s s , N.Y. ( 1 9 6 6 ) , p. 273. 11. D.M. Blow, J . J . B i r k t o f t , and B.S. H a r t l e y , N a t u r e 221, 337 (1 9 6 9 ) . 12. W.N. Lipscomb, J.A. H a r t s u c k , F.A. Q u i o c h o , and G.N. Reeke, J r . , P r o c . N a t l . Acad. S c i . U.S.A. 64, 28 ( 1 9 6 9 ) . 13. b.E. K o s h l a n d , J r . , Adv. Enzymol. 22_, 45 (1 9 6 0 ) ; D.E. K o s h l a n d , J r . , J . T h e o r e t . B i o l . 2, 75 ( 1 9 6 2 ) . 14. D.R. Storm and D.E. K o s h l a n d , J r . , P r o c . N a t l . Acad. S c i . U.S.A. 66, 445 ( 1 9 7 0 ) . - 1 0 9 -15. T.C. B r u i c e , A. Brown, and D.O. H a r r i s , P r o c . N a t l . A cad. S c i . U.S.A. 67_, (197 1 ) . 16. C.C.F. B l a k e , G.A. M a i r , A.C.T. N o r t h , D.C. P h i l l i p s , and V.R. Sarma, P r o c . Roy. Soc. BI67, 365 ( 1 9 6 7 ) . 17. B.D. Sy k e s , S.L. P a t t , and D. D o l p h i n , C o l d S p r i n g H a r b o r Symp. Quant. B i o l . 36, 29 ( 1 9 7 1 ) . 18. F.A. Quiocho and W.N. Lipscomb, Adv. P r o t e i n Chem. 25_, 1 (1 9 7 1 ) . 19. W.J. O ' S u l l i v a n and M. Cohn, J . B i o l . Chem. 241_, 3116 ( 1 9 6 6 ) . 20. R. Freeman and S. W i t t e k o e k , J . Mag. Res. 1_, 238 (1969), 21. A.G. M a r s h a l l , P.G. Sc h m i d t , and B.D. S y k e s , B i o c h e m i s t r y ]_}_, 3875 ( 1 9 7 2 ) ; A.G. M a r s h a l l and L.G. Werbelow, J . Amer. Chem. S o c , 95, 5132(1973). 22. R. B u r t o n , C.W.M. G r a n t , and L.D. H a l l , Can. J . Chem. 50, 497 ( 1 9 7 2 ) . 23. S. Meiboom and D. G i l l , Rev. S c i , I n s t r u m . 2 £ , 668 ( 1 9 5 8 ) . 24. H.Y. C a r r and E.M. Puree! 1, Phys. Rev. 94_, 630 (195 4 ) . 25. B.D. S y k e s , P.G. Schmidt and G.R. S t a r k , J . B i o l . Chem. 245, 1180 (19 7 0 ) . 26. B.D. S y k e s , and C. P a r r a v a n o , J . B i o l . Chem. 244, 3900 ( 1 9 6 9 ) . 27. A.G. M a r s h a l l and L.G. Werbelow, J . Mag. Res., i n p r e s s . 28. R.P. Haugland and L. S t r y e r , i n C o n f o r m a t i o n o f B i o p o l y m e r s , V o l . 1, ed. G.N. Ramachandran (Academic P r e s s , N.Y., 1967), p. 321. 29. T. Inagami and H. Hatano, J . B i o l . Chem. 244, 1176 (196 9 ) . - n o -30. R.M. S t r o u d , L.M. Kay, and R.E. D i c k e r s o n , C o l d S p r i n g H a r b o r Symp. Quant. B i o l . 36, 125 ( 1 9 7 1 ) . 31. B.M. Sanborn and G.E. H e i n , B i o c h e m i s t r y 7_, 3616 ( 1 9 6 8 ) ; B i o c h i m . B i o p h y s . A c t a 139, 524 ( 1 9 6 7 ) . 32. M.L. Bender, R.L. Van E t t e n , and G.A. Clowes, J . Amer. Chem. Soc. 88, 2319 ( 1 9 6 6 ) . 33. F. Seydoux, G. C o u t o u l y , and J . Yon, B i o c h e m i s t r y 1_0, 2284 ( 1 9 7 1 ) . 34. T. Yamamoto and N. I z u m i y a , A r c h . Biochem. B i o p h y s . 120, 497 ( 1 9 6 7 ) . I - I l l -CHAPTER V M a g n e t i c Resonance S t u d i e s o f S e r i n e - 1 9 5 - S p i n - L a b e l e d T r y p s i n A. I n t r o d u c t i o n A s p i n l a b e l i s a s y n t h e t i c p a r a m a g n e t i c o r g a n i c f r e e r a d i c a l , u s u a l l y h a v i n g a m o l e c u l a r s t r u c t u r e a n d / o r c h e m i c a l r e a c t i v i t y t h a t r e s u l t s i n i t s a t t a c h m e n t o r i n c o r p o r a t i o n a t some p a r t i c u l a r s i t e , c o v a l e n t l y o r n o n c o v a l e n t l y i n a b i o l o g i c a l m a c r o m o l e c u l e , o r assemblage o f m a c r o m o l e c u l e s . T h i s t y p e o f probe was f i r s t i n t r o d u c e d 1 2 by M c C o n n e l l and was s u c c e s s f u l l y a p p l i e d t o s t u d i e s o f membranes 3 as w e l l as s t u d i e s o f t h e c o n f o r m a t i o n a l changes i n m a c r o m o l e c u l e s . 4 5 S e v e r a l e x c e l l e n t r e v i e w a r t i c l e s a r e now a v a i l a b l e ' . R e c e n t l y , s e v e r a l p a p e r s have e x p l o i t e d t h e l a r g e m a g n e t i c moment a s s o c i a t e d w i t h an u n p a i r e d e l e c t r o n , t o enhance t h e d i p o l a r r e l a x a t i o n o o f n u c l e i a t d i s t a n c e s o f as much as 10 A away. Thus the d e t e c t i o n o f t h e d i p o l a r i n t e r a c t i o n between t h e n i t r o x i d e s p i n l a b e l and the n u c l e u s o f i n t e r e s t p e r m i t s t h e e s t i m a t e o f t h e d i s t a n c e s o v e r a r e l a t i v e l y l a r g e r a n g e . The e s t i m a t e o f su c h a l a r g e d i s t a n c e can n o t u s u a l l y be o b t a i n e d t h r o u g h t he weak d i p o l a r i n t e r a c t i o n between a p a i r o f d i s t a n t n u c l e i . M i l d v a n and W e i n e r ^ have used a s p i n - l a b e l e d a n a l o g o f N i c o t i n a m i d e A d e n i n e D i n u c l e o t i d e t o p e r t u r b the l i n e w i d t h o f e t h a n o l and some o t h e r - 112 -s u b s t r a t e s i n L i v e r A l c o h o l Dehydrogenase (LAD). The r e l a t i v e p o s i t i o n on LAD o f the a p p r o p r i a t e coenzymes and the s u b s t r a t e s may then be r e c o n s t r u c t e d . E x p e r i m e n t s u s i n g b o t h p a r a m a g n e t i c metal i o n s and n i t r o x i d e s p i n l a b e l s t o enhance n u c l e a r r e l a x a t i o n r a t e s and t o e s t i m a t e i n t r a m o l e c u l a r d i s t a n c e s i n enzyme complexes i n s o l u t i o n have a l s o 7 8 9 been d e s c r i b e d ' ' . o R e c e n t l y , d i s t a n c e s o f up t o 20 A between e i t h e r bound s a c c h a r i d e s o r H i s t i d i n e - 1 5 , and T r yptophan-123 on lysozyme i n s o l u t i o n have been e s t i m a t e s f r o m the s p i n - l a b e l i n d u c e d l i n e b r o a d e n i n g o f t h e p r o t o n o f i n t e r e s t by Wien e t a l 1 0 . In the l a s t c h a p t e r o f t h i s t h e s i s , t h i s p o w e r f u l s p i n l a b e l t e c h n i q u e w i l l be a p p l i e d t o h e l p d e m o n s t r a t e t h e e x i s t e n c e o f the s e c o n d a r y b i n d i n g s i t e w h ich had been s u s p e c t e d from e a r l i e r i n d i r e c t 11 12 13 e v i d e n c e ' ' and f r o m the UV s t u d i e s ( c h a p t e r I I I ) i n t h i s t h e s i s . o S i n c e t h e d i a m e t e r o f t r y p s i n i s o n l y a b o u t 40 t o 50 A, even i f the d i p o l a r r e l a x a t i o n i n d u c e d by t h e u n p a i r e d e l e c t r o n o f t h e n i t r o x i d e s p i n l a b e l i s n o t d e t e c t a b l e , t h e r e s u l t w i l l a t l e a s t l i m i t t he number o f p o s s i b l e l o c a t i o n s f o r the s e c o n d a r y s i t e . I f the l i n e b r o a d e n i n g i n d u c e d by the n i t r o x i d e s p i n l a b e l i s i n d e e d o b s e r v a b l e , t h e n d i s t a n c e s between each i n h i b i t o r s and the n i t r o x i d e o f t h e s p i n l a b e l a t S e r i n e -195, d e t e r m i n e d by n u c l e a r r e l a x a t i o n measurements, and w i t h the h e l p o f the X-ray t h r e e d i m e n s i o n a l s t r u s t u r e o f t r y p s i n s h o u l d e n a b l e one t o make a r e s o n a b l e guess as t o where the s e c o n d a r y s i t e on t r y p s i n c o u l d be. I t i s known t h a t the ESR s p e c t r a o f s p i n l e b e l s a r e v e r y s e n s i t i v e - 113 -4 14 t o t h e r a t e a t w h i c h t h e l a b e l i s a b l e t o r e o r i e n t r o t a t i o n a l l y ' , t h a t i s , t h e ESR s p e c t r u m o f the l a b e l w i l l r e f l e c t the degree o f r o t a t i o n a l m o b i l i t y a l l o w e d i n the e n v i r o m e n t o f t h e l a b e l . S i n c e t h e n i t r o x i d e o f t h e s p i n l a b e l was a t t a c h e d t o t h e 15 S e r i n e - 1 9 5 a t the a c t i v e s i t e , one w o uld e x p e c t t h a t the f r e e r a d i c a l a t t h e a c t i v e s i t e w i l l p r o v i d e a good probe t o m o n i t o r t h e c o n f o r m a t i o n a l changes a t t h e a c t i v e s i t e . "Thus i t s h o u l d be p o s s i b l e ++ t o o b s e r v e any c o n f o r m a t i o n a l changes i n d u c e d upon a d d i n g the Ca o r M n + + i o n , as w e l l as t o examine t h e i n t e r a c t i o n between t h e s p i n l a b e l a t t a c h e d t o t h e S e r i n e - 1 9 5 and t h e i n h i b i t o r s bound t o the s e c o n d a r y b i n d i n g s i t e s , by m o n i t o r i n g the ESR s p e c t r u m o f t h e f r e e r a d i c a l i n t h e a c t i v e s i t e . F i n a l l y , i n o r d e r t o g e t r i d o f the r e p o r t e d u n d a s i r e d a u t o p r o -t e o l y s i s c a u s e d by t h e p r e s e n c e o f t r a c e amount o f t r y p s i n i n the s p i n l a b e l e d t r y p s i n , a p r e p a r a t i o n o f a c t i v e t r y p s i n - f r e e s p i n l a b e l e d t r y p s i n was d e s i g n e d and c a r r i e d o u t as d e s c r i b e d i n t h e e x p e r i m e n t a l s e c t i o n o f t h i s c h a p t e r . B. E x p e r i m e n t a l 1. M a t e r i a l s S p i n l a b e l 4 - H y d r o x y - 2 . 2 . 6 . 6 . - t e t r a m e t h y l p i p e r i d i n o o x y l m o n o e t h y l p h o s p h o r o f l u o r i d a t e ( e s t e r ) was o b t a i n e d from S y n v a r . Sephadex G-25, G-50 and i o n exchange Sp-C50 were from P h a r m a c i a F i n e C h e m i c a l s . The r e s t o f t h e c h e m i c a l s a r e a l l o b t a i n e d from the same s o u r c e as i n p r e v i o u s c h a p t e r s . 4 - 114 -2. P r e l i m i n a r y p r e p a r a t i o n o f g e l f i l t r a t i o n columns Sephadex G-50 was h y d r a t e d i n 0.05 M T P J S - C h l o r i d e b u f f e r (pH = 7.1), c o n t a i n i n g 0.02 M C a c l g , a t room t e m p a r a t u r e f o r 12 h o urs w i t h f r e q u e n t d e c a n t a t i o n s t o remove f i n e m a t e r i a l , and d e g a s s e d f o r t h r e e hours on a w a t e r a s p i r a t o r . I t was then poured i n t o a column (5.0 x 80 cm) a n d - o p e r a t e d w i t h the s o l v e n t r e s e r v o i r t y p i c a l l y below t h e head o f t h e column t o a v o i d p a c k i n g . About two t o t h r e e bed volumes o f b u f f e r was e l u t e d t h r o u g h the column t o a c h i e v e e q u i l i b r i u m The same p r o c e d u r e was employed f o r t h e p r e p a r a t i o n o f Sephadex G-25 (5.0 x 85 cm), Sp-Sephadex C-50 columns (2.6 x 5 0 ) . 3. P r e p a r a t i o n o f a c t i v e t r y p s i n - f r e e , s p i n l a b e l e d t r y p s i n T r y p s i n was c o v a l e n t e l y l a b e l e d a t S e r i n e - 1 9 5 w i t h s p i n l a b e l 1 g i n h i b i t o r by a m o d i f i c a t i o n o f the method d e s c r i b e d by B e r l i n e r 25 mg o f the s p i n l a b e l i n h i b i t o r ( 4 - H y d r o x y - 2 , 2 , 6 , 6 , - t e t r a methyl p i p e r i d i n o o x y l m o n o e t h y l p h o s p h o r o f l u o r i d a t e ( e s t e r ) ) f r o m S y n v a r was d i s s o l v e d c o m p l e t e l y i n 1 ml d i o x a n e . H a l f o f the s p i n l a b e l was added to a s o l u t i o n c o n t a i n i n g 20 ml o f 0.05 M T r i s -C h l o r i d e b u f f e r (pH = 7.7, 0.02 M C a C l J and 5 ml o f d i o x a n e . A f t e r a t 5° C. C H 3 - 115 -t h e s e two s o l u t i o n s were c o m p l e t e l y mixed, 250 mg o f t r y p s i n were added, t h e pH was a d j u s t e d t o 7.8 w i t h 1 M NaOH, and t h e s o l u t i o n were s t i r r e d s l o w l y . About 30 m i n u t e s l a t e r , t h e r e s t o f the s p i n l a b e l was p o u r e d i n t o the r e a c t i o n m i x t u r e . The r e a c t i o n was a l l o w e d to p r o c e d f o r about a n o t h e r two hours a t room t e m p e r a t u r e . The o r g a n i c s o l v e n t s e r v e s t o h e l p d i s s o l v i n g t h e s p i n l a b e l as d e s c r i b e d l fi i n B e r l i n e r ' s work . I t was a l s o shown t h a t 50% (V/V) d i o x a n e does n o t e f f e c t enzyme a c t i v i t y ^ . The l a b e l e d enzyme was then p a s s e d t h r o u g h a column (5.0 x 85 cm) o f Sephadex G-25 e q u i l i b r a t e d w i t h 0.1 M NaCl and 0.01 M a c e t i c a c i d , pH 3.3 a t 5° C. The l a s t few tubes c o n t a i n i n g p r o t e i n were c h e c k e d by ESR t o e n s u r e c o m p l e t e removal o f f r e e u n r e a c t e d s p i n l a b e l . Then f r a c t i o n s o f the e f f l u e n t w h ich c o n t a i n e d p r o t e i n w i t h o u t f r e e s p i n l a b e l were p o o l e d t o g e t h e r and 100 mg soybean t r y p s i n i n h i b i t o r was added, t o p r e v e n t a t t a c k on s p i n - l a b e l t r y p s i n by ( u n r e a c t e d ) a c t i v e t r y p s i n . A f t e r 7 volumes exchange w i t h t h e 0.05 M TRIS-C1 b u f f e r (pH = 7.1, C a C l g , 0.02 M) by c o n t i n u o u s u l t r a f i l t r a t i o n (UM-10 membrane from Ami con C o r p . ) , the s o l u t i o n was c o n c e n t r a t e d down t o 40 ml. A s m a l l amount o f t h e i n s o l u b l e m a t e r i a l which formed d u r i n g c o n c e r n t r a t i o n was removed by c e n t r i f u g a t i o n and the s u p e r n a t a n t s o l u t i o n was a p p l i e d t o a column (5.0 x 80 cm) o f sephadex G-50 which had been e q u i l i b r a t e d w i t h the b u f f e r b e i n g exchanged b e f o r e . The column was e l u t e d a t a f l o w r a t e o f 30 ml p e r h o u r and the e l u a t e was c o l l e c t e d i n 8 ml f r a c t i o n s ( F i g u r e 5.1). The s e c o n d peak was p o o l e d t o g e t h e r and was c o n c e n t r a t e d down t o 40 ml by u l t r a f i l t r a t i o n . The c o n c e n t r a t e was examined by ESR a g a i n t o i d e n t i f y t h e compound. In F i g u r e 5.1, the f i r s t peak - 116 -r e p r e s e n t s t r y p s i n - soybean t r y p s i n i n h i b i t o r complex, which i s I o e x c l u d e d from t h e g e l due t o i t s h i g h m o l e c u l a r w e i g h t (45,000) , and the t h i r d peak r e p r e s e n t s a u t o l y z e d p o l y p e p t i d e . T h i s p r o c e s s removes a l m o s t a l l o f the u n r e a c t e d a c t i v e t r y p s i n i n t h e f o r m o f STI - T complex, b e c a u s e t h e d i s s o c i a t i o n c o n s t a n t t h e t r y p s i n --8 19 soybean t r y p s i n i n h i b i t o r i s s m a l l e r t h a n 10 a t PH 7.1 40 ml o f the c o n c e n t r a t e from the 2nd peak was then a p p l i e d t o a Sp-Sephadex C-50 column (2.6 x 5 0 ) , t o g e t r i d o f the i n a c t i v e 20 t r y p s i n . A t y p i c a l e l u t i o n d i a g r a m i s shown i n F i g u r e 5.2. The a and 3 t r y p s i n f r a c t i o n s were p o o l e d s e p e r a t e l y and d i a l y z e d a g a i n s t 0.01 M a c e t i c a c i d i n the c o l d . A s m a l l f r a c t i o n o f each p o o l e d and the d i a l i z e d s o l u t i o n was c o n c e n t r a t e d and ESR s p e c t r a were- o b t a i n e d - ( F i g u r e 5-.3)-. The r e s t o f t h e d i a l y z e d s o l u t i o n was-l y o p h o l i z e d , and the l y o p h o l i z e d sample was a l s o examined by ESR ( F i g u r e 5 . 4 ) . The s p i n l a b e l e d e - t r y p s i n ( b e f o r e f r e e z e d r y i n g ) was l e f t a t room t e m p e r a t u r e (25° C) pH = 7.1 and t h e r e were no changes i n the ESR s p e c t r u m f o r a t l e a s t 3 days ( F i g u r e 5.5). Thus we can be c o n f i d e n t t h a t t h e r e was no a c t i v e t r y p s i n i n t h i s s p i n - l a b e l e d t r y p s i n p r e p a r a t i o n . Because t h e i s o e l e c t r i c p o i n t o f s o y b e a n 21 t r y p s i n i n h i b i t o r ( S T I ) i s 4.5 , STI w i l l n o t b i n d t o a c a t i o n i o n - e x c h a n g e r a t n e u t r a l pH. Hence the e x c e s s STI s h o u l d be e l u t e d o u t f i r s t ( a l o n g w i t h i n a c t i v e t r y p s i n ) from t h e SP - C 50 column ( F i g u r e 5.2). The b r o a d ESR s i g n a l o f s p i n - l a b e l e d g - t r y p s i n s h a r p e n s t o a narrow s i g n a l a f t e r a d d i n g commercial t r y p s i n ( F i g u r e 5 . 6 ) , as o b s e r v e d by B e r l i n e r ^ . - 117 -F i g u r e 5.1. S e p a r a t i o n o f t r y p s i n - Soybean t r y p s i n i n h i b i t o r ( S T I ) complex from a m i x t u r e o f s p i n l a b e l e d t r y p s i n , t r y p s i n - S T I complex and STI by chromatograph on a column (5.0 x 80 cm) o f sephadex G-50, i n 0.05 M t r i s - c h l o r i d e b u f f e r , pH 7.1, 0.02 M C a C 1 2 a t 4 ° C. The f l o w r a t e was 30 ml p e r hour; 8 ml were c o l l e c t e d p e r t u b e . T U R F M I I M R P R - 119 -F i g u r e 5.2. S e p a r a t i o n o f a and B s p i n l a b e l e d t r y p s i n from a m i x t u r e o f a and 3 s p i n l a b e l e d t r y p s i n , S T I , and i n a c t i v e ( s p i n l a b e l e d ) t r y p s i n by i o n exchange c h r o m o r t o g r a p h y on a column (2.6 x 50 cm) o f Sp-Sephadex C-50 i n 0.05 M T r i s - C h l o r i d e b u f f e r , PH 7.1, 0.02 M C a C l 2 , a t 4° C. The f l o w r a t e was 15 t o 20 ml p e r h o u r ; 8.5 ml were c o l l e c t e d p e r t u b e . 1.5 - 121 -F i g u r e 5.3- ESR spectrum o f f r e e s p i n l a b e l , s p i n l a b e l e d a and 3 t r y s p i n . A. ESR s p e c t r u m o f f r e e s p i n l a b e l (4 - Hydroxy - 2, 2, 6, 6, -t e t r a m e t h y l p i p e r i d i n o o x y l m o n o e t h y l p h o s p h o r o f l u o r i d a t e e s t e r ) i n 0.05 M T R I S - C h l o r i d e b u f f e r , PH = 7.1., B and C. ESR s p e c t r a o f s p i n l a b e l e d a - t r y p s i n ( s p e c t r u m B) and 3 - t r y p s i n ( s p e c t r u m C) i n 0.05 M T R I S - C h l o r i d e b u f f e r PH = 7.1 0.02 M C a C l g b e f o r e f r e e z e d r y i n g . The s p e c t r u m were unchanged even a f t e r t h e s o l u t i o n s were s e p a r a t e l y d i a l i z e d a g a i n s t 0.01 M a c e t i c a c i d (PH 3.3) f o r 24 hours a t 4° C. E-3 ESR SPECTROMETER SETTINGS. F i e l d S e t : 3380 G M o d u l a t i o n A m p l i t u d e : 0.5 G Scan time 16 min R e c e i v e r G a i n Scan range : 100 G - 122 -- 123 -F i g u r e 5.4. A c o m p a r i s o n o f the ESR s p e c t r a o f s p i n l e b e l e d - t r y p s i n b e f o r e and a f t e r f r e e z e d r y i n g . A. ESR s p e c t r a o f s p i n - l a b e l e d - g t r y p s i n i n 0.05 M T r i s - C h l o r i d e b u f f e r , PH 7.1, 0.02 M C a C l g . B. A s m a l l p e r c e n t a g e o f s p i n - l a b e l e d - g t r y p s i n was d e n a t u r e d by the f r e e z e d r y i n g p r o c e s s . The d e n a t u r a t i o n was i n d i c a t e d by the apperance o f a s h a r p s i g n a l a t the h i g h f i e l d r e s o n a n c e l i n e . C. The ESR s p e c t r a o f s p i n - l a b e l e d - g t r y p s i n which has been i n c u b a t e d i n t h e NMR probe (40° C) f o r 30 min : no n o t i c e a b l e change i s o b s e r v e s . T h i s e n s u r e s t h a t d e n a t u r a t i o n d u r i n g the NMR e x p e r i m e n t can be n e g l e c t e d . - 124 -- 125 -F i g u r e 5.5. The s t a b i l i t y o f the a c t i v e t r y p s i n - f r e e , s p i n - l a b e l e d - t r y p s i n i s shown by the i n v a r i a n t ESR s p e c t r u m o f s p i n - l a b e l e d - 3 t r y p s i n i n T r i s - C h l o r i d e b u f f e r PH 7.1, 0.02M C a C l 2 a t room t e m p e r a t u r e (20-25°C ) f o r t h r e e d a y s . A. i m m e d i a t e l y a f t e r b e i n g c o n c e n t r a t e d from Amicon c e l l . B. 2 days l a t e r . C. 3 days l a t e r . - 120 -- 127 -F i g u r e 5.6. The h y d r o l y s i s o f s p i n - l a b e l e d t r y p s i n on a d d i t i o n o f a s m a l l amount o f commercial ( a c t i v e ) t r y p s i n i n 0.05 M T r i s - C h l o r i d e b u f f e r PH 7.1, 0.02 M C a C l 2 . A. 30 min a f t e r t h e a d d i t i o n o f t r y p s i n . B. 12 hours a f t e r t h e a d d i t i o n o f t r y p s i n . C. 3 days a f t e r t h e a d d i t i o n o f t r y p s i n . - 128 -- 129 -F i g u r e 5.7 A. E.S.R. s p e c t r a o f 5 x 1 0 ~ 4 M n + + s o l u t i o n (0.05 M T R I S - C h l o r i d e b u f f e r Ph = 7.1) i n t h e p r e s e n c e o f 1.26 x 1 0 " 4 M / £ . S.L. TRYPSIN. e x c e p t t h e E.S.R. s p e c t r a o f t h e s p i n l a b e l t r y p s i n was s u p e r i m p o s e d ++ on t o p o f t h e c e n t e r o f t h e Mn sp e c t r u m . B. The e x p a n s i o n o f t h e c e n t e r p o r t i o n o f A. The E.S.R. s i g n a l o f the s p i n l a b e l l e d t r y p s i n i s now v i s i b l e , and no o b v i o u s change c an be d e t e c t e d . C. E.S.R. s p e c t r u m o f 1.26 x 1 0 " 4 M/L S L — T R Y P S I N i n t h e p r e s e n c e o f 0.025 M p - m e t h o x y l . p h e n y l G u a n i d i n e - H C l (good i n h i b i t o r ) and 0.02 M C a C l g . D. E.S.R. sp e c t r u m o f t h e 1.26 x 1 0 " 4 S L - T R Y P S I N i n t h e p r e s e n c e o f 2.4 x 1 0 " 3 MTAME and 0.02 M C a C l 9 . - 130 -- 131 -4. Method S p i n l a b e l e d t r y p s i n s t o c k s o l u t i o n . 21 mg o f s p i n l a b e l e d t r y p s i n was d i s s o l v e d i n 2 ml o f DgO B o r a t e b u f f e r (pDH = 8 . 1 ) , c o n t a i n i n g 0.02 M CaClg- The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n . The c o n c e n t r a t i o n o f t h e p r o t e i n i n t h e s u p e r n a t a n t was d e t e r m i n e d on a C a r l - Z e i s s PMO-II by m e a s u r i n g the a b s o r p t i o n a t 280 nm u s i n g an e x t i n c t i o n c o e f f i c i e n t o f 0.651 mg ml~^ (OD u n i t ) " ^ 22 and assuming a m o l e c u l a r w e i g h t o f 24,000 . T h i s s o l u t i o n was s t o r e d i n i c e b a t h f o r l a t e r u s e . Samples f o r NMR measurement were p r e p a r e d by a d d i n g 0.2 ml o f the s p i n - l a b e l e d t r y p s i n s t o c k s o l u t i o n and 0.2 ml o f the i n h i b i t o r s t o c k s o l u t i o n . B e f o r e i n s e r t i o n i n t o t he NMR p r o b e , t h e sample was prewarmed i n a c o n s t a n t t e m p e r a t u r e b a t h a t 30° C f o r 3 m i n u t e s . The NMR p r o b e t e m p e r a t u r e was m o n i t o r e d t h r o u g h a t h e r m i s t e r (YS1 Model 42SC Te l e - T h e r m o m e t e r ) i n a NMR tube and was c o n t r o l l e d t o be 30 +_ 1 ° C by r u n n i n g Ng gas t h r o u g h a c o o l i n g c o i l immersed i n a w a t e r - a n d - i c e m i x t u r e . The V a r i a n t e m p e r a t u r e -c o n t r o l u n i t and the c o o l e n t N^ U) were n o t used b e c a u s e the t e m p e r a t u r e v a r i a t i o n was t oo b i g +_ 3 - 5 ° C and t h e h e a t c a p a c i t y o f NgU) i s t o o s m a l l t h a t i t i s n e c e s s a r y t o r e f i l l t h e t h e r m o f l a s k e v e r y hou r . The r e f i l l i n g p r o c e s s u s u a l l y w i l l d i s t u r b the t e m p e r a t u r e a t t h e probe as w e l l as t h e s t a b i l i t y o f t h e s p e c t r o m e t e r . A l l t h e l i n e w i d t h s t u d i e s were done on a v a r i a n XL-100-15 NMR s p e c t r o m e t e r w i t h a FT-8K on l i n e computer. The e x p a n s i o n o f t h e i n t e r e s t i n g p o r t i o n o f the s p e c t r u m can be done by the t e l e t y p e c o n n e c t e d t o t h e computer. The f i e l d h o m o g e neity was o p t i m i z e d by - 132 -f i r s t l o c k i n g on t h e ^H r e s o n a n c e o f HDO, the n s w i t c h i n g t o the 2 H l o c k u s i n g t h e DgO peak. F i e l d homogeneity c o n t r o l was l e f t u n d i s t u r b e d w h i l e the p u l s e d F.T. NMR measurement was c a r r i e d o u t . The pH o f s o l u t i o n s was measured a t room t e m p e r a t u r e w i t h a Copenhagen R a d i o - m e t e r e q u i p p e d w i t h a Beckman 39030 c o m b i n a t i o n e l e c t r o d e , A l l r e p o r t e d pH v a l u e s have been s u b j e c t e d t o t h e 23 c o r r e c t i o n (pH = meter r e a d i n g - 0.4) f o r s o l u t i o n s i n DgO. C. T h e o r y The c o n d i t i o n s f o r f a s t exchange can be f o u n d i n C h a p t e r 1 ca s e B ( d ) ; 1 /l x ^ » ^ > E I . 1 - d - ) C T » A W 2 (5.1) T E I YEI EI where dr-) = ( J - + - L - ) (5.2) '2 t l '2B '2M Her e , T ^ J i s t h e l i f e t i me f o r the i n h i b i t o r bound t o t h e enzyme; ^ 2 ^ E I A N C * ^2A a r e ^ e t r a n s v e r s e n u c l e a r r e l a x a t i o n t imes o f t h e p r o t o n o f the i n h i b i t o r f o r E I complex and f r e e i n h i b i t o r r e s p e c t i v e l y . Tgg and T g ^ ar e c o n t r i b u t i o n s from n u c l e u s - n u c l e u s d i p o l e - d i p o l e i n t e r a c t i o n and e l e c t r o n - n u c l e u s d i p o l e - d i p o l e i n t e r a c t i o n f o r E I complex r e s p e c t i v e l y . AW^J i s the d i f f e r e n c e o f the r e s o n a n c e f r e q u e n c i e s o f the bound and f r e e i n h i b i t o r s . - 1 3 3 -Under the above c o n d i t i o n s o f f a s t exchange, the o b s e r v e d t r a n s v e r s e r e l a x a t i o n time w i l l be f = r~+ V r - - ! 1 - } ( 5- 3 ) '2 12A L i 12B '2M where f £ I i s the f r a c t i o n o f i n h i b i t o r s bound t o the enzyme ; f ^ = 1 - f E I ; a l s o f A » f E I thus f ^ = 1 . From C h a p t e r I e q u a t i o n s ( 1 . 1 8 ) the t r a n s v e r s e r e l a x a t i o n i n d u c e d by t h e u n p a i r e d e l e c t r o n o f s p i n l a b e l s can be d e s c r i b e d as ! i S ( S + l ) Y T g V 3 T 1 3 T TOM 15 6 [ 4 T C r , . .,2 2 ' . " 2 2 J 2M r 1 + W TT 1 + '/ IT I c s c However, i n the p r e s e n t e x p e r i m e n t ; the u n p a i r e d e l e c t r o n o f t h e n i t r o x i d e and the p r o t o n o f the i n h i b i t o r , a r e w e l l s e p a r a t e d i n t h e EI complex. Thus we may o m i t the s e c o n d term c o r r e s p o n d i n g t o i s o t r o p i c s p i n - e x c h a n g e i n t e r a c t i o n . F u r t h e r m o r e , when the i n h i b i t o r b i n d s t o t h e enzyme T c i n c r e a s e s and t o a good 2 2 a p p r o x i m a t i o n W T >> 1. Under t h e s e c o n d i t i o n s , the n u c l e a r t r a n s v e r s e r e l a x a t i o n t ime can be s i m p l i f i e d t o ! S(S + l ) Y 2 g V 3T ' = i [ 4 + c ] ( 5 . 5 ) ' 2M 15r c 1 + \Cxt I c s o , i f Tgi^i and T c a r e known, the d i s t a n c e between the u n p a i r e d e l e c t r o n and t h e p r o t o n o f i n t e r e s t can be d e t e r m i n e d . As w i l l be d i s c u s s e d l a t e r , T £ i n t h i s e q u a t i o n i s e s s e n t i a l l y - 134 -eq u a l t o the r o t a t i o n a l c o r r e l a t i o n t i m e o f the enzyme . A c o n v i e n i e n t e x p r e s s i o n f o r r ( i n A) i n terms o f T 2 M ( i n s e c ) i s 3T r ( A ) = 4 8 5 [ T 2 M ( 4 T + ^ r ? ) ) y 6 (5.6) 1 + W j x c O OA where ^ = 1 . 6 x 1 0 ° s e c and Wj = 100 MHz D. A n a l y s i s o f d a t a I . D e t e r m i n a t i o n o f T 2 M and r In t h e a b s e n c e and p r e s e n c e o f the s p i n - l a b e l e d enzyme, t h e o b s e r v e d t r a n s v e r s e r e l a x a t i o n t i m e f o r the i n h i b i t o r w i l l be (f) = T ^ r - <5-7> '2 .2A 12H T 2 A T 2 A T 2 V T 2 C !2H* b i *2B l2M where f A = 1 i f [ I ] » [E I ] f ^ j i s t h e f r a c t i o n o f bound i n h i b i t o r s , can be c a l c u l a t e d f r o m K T ( C h a p t e r I V ) . j^— i s t h e t r a n s v e r s e r e l a x a t i o n time o f 1 *2A f r e e i n h i b i t o r s . where j^—, y ^ — , v^ -> r e p r e s e n t t h e c o n t r i b u t i o n from v i s c o s i t y '2V 2C '2H change by the p r e s e n c e o f enzyme, the c o n t a c t i n t e r a c t i o n between the u n p a i r e d e l e c t r o n on th e s p i n l a b e l and the Drotons o f i n h i b i t o r .and t h e f i e l d irahomogeneity a t the time o f measurement, r e s p e c t i v e l y . - 135 -The r e l a x a t i o n time f o r an i n t e r n a l s t a n d a r d (which w i l l n o t b i n d t o t h e enzyme) i n t h e a b s e n c e and p r e s e n c e o f enzyme w i l l be '2 s '2A *2H (f-)* = (J-)' + 1 . + -L+ ( 1 )'. (5.10) '2 s '2A '2V 12C '2H By u s i n g a m u l t i c h a n n e l p u l s e NMR s p e c t r o m e t e r , the l i n e w i d t h o f b o t h t h e i n t e r n a l s t a n d a r d and t h e i n h i b i t o r can be measured a t t h e same time ; thus 1 = ( 1 )• and ( ^ = ( y U ) ' 12H '2H 2H 12H. • Ncvr, t h e - t e r m f^— can e a s i l y b e - f o u n d - a s f o l l o w s '2M *2M T E I ( '2 ' 2 s '2 ' 2 s ) 12B = T 1 - x ( N e t b r o a d e n i n g ) - . (5.11) T E I 12B Here ( i - ) * , ( i - ) * , (j-) and OF-) can be d e t e r m i n e d e x p e r i m e n t a l l y 2 2 2 2 and i^r—) has been d e t e r m i n e d p r e v i o u s l y i n C h a p t e r 4 . ( 1 / T ? M ) 12B v a l u e s f o r each o f the i n h i b i t o r s a r e g i v e n i n t a b l e 5.1 (30°C) and t a b l e 5.2 ( 4 0 ° C ) . With t h e a s s u m p t i o n s ( t o be p r o v e d l a t e r ) t h a t t h e f a s t exchange l i m i t i s v a l i d and T c i s dominated by x r , t h e d i s t a n c e between t h e f r e e r a d i c a l and the methyl group o f each i n h i b i t o r s can be c a l c u l a t e d by s u b s t i t u t i n g ( y ^ — ) f o r each i n h i b i t o r s •2M i n t o e q u a t i o n ( 5 . 6 ) . The r e s u l t s can be f o u n d i n the l a s t column o f t a b l e 5.1. 136 -2. x i s dominates by x As i n C h a p t e r 1 e q u a t i o n ( 1 . 2 0 ) , the c o r r e l a t i o n t i m e , x , w h i c h c h a r a c t e r i z e s the r a t e p r o c e s s t h a t m o d u l a t e s the d i p o l a r i n t e r a c t i o n s i s g i v e n by 1_ = 1_ + i _ + J _ ( 5 i l 2 ) T c T r T s T E I where x ^ ; r o t a t i o n a l c o r r e l a t i o n t i m e , x g ; e l e c t r o n i c s p i n l a t t i c e r e l a x a t i o n t i m e , x ^ j ; r e s i d e n c e time f o r the i n h i b i t o r s on the enzyme. The c o r r e l a t i o n time i s d e t e r m i n e d by the f a s t e s t r a t e p r o c e s s ; t h a t i s , which e v e r time i s s h o r t e s t - x . x o r x F T , S i n c e t h e m o l e c u l a r w e i g h t o f t r y p s i n (23.800) i s about the same as ch e m o t r y p s i n ( 23 .200) , we would e x p e c t t h e r o t a t i o n a l c o r r e l a t i o n time f o r t r y p s i n w i l l be a b o u t the same as t h a t o f c h e m o t r y p s i n , _ Q i . e , 1.6 x 10" s e c . The exchange l i f e t i m e , x ^ j , may be e s t i m a t e d from Kj and k-j (t h e r a t e o f f o r m a t i o n s o f the enzyme i n h i b i t o r complex) as f o l l o w i n g ; 9 -1 2 5 s i n c e the t y p i c a l v a l u e s f o r k-j i s s m a l l e r o r e q u a l t o 10 s e c , and the Ka f o r t h e i n h i b i t o r s s t u d i e d h e r e a r e l a r g e r o r e q u a l t o 1 0 2 M" 1 ( C h a p t e r I I I ) k i = = - ^ t = 1 0 7 s e c " 1 (5 .13) 10 and — = k , (5 .14) T E I "' thus Xpj s h o u l d e i t h e r c l o s e t o o r l a r g e r t h a n 10~ 7 , and t h i s i s a b o u t one o r d e r o f magnitude s l o w e r than t h e r o t a t i o n a l c o r r e l a t i o n - 137 --8 time (10 ) . C l e a r l y I / T ^ J can n o t dominate t h e r a t e p r o c e s s . The e l e c t r o n i c r e l a x a t i o n t i m e , x s , f o r Fremy's s a l t a 7 26 s o l i d n i t r o x i d e s p e c i e s ) , i s 3.4 x 10" s e c . A l s o a n a l y s i s o f the s a t u r a t i o n b e h a v i o r o f 3 - S L - H i s - 1 5 - l y s o z y m e has been r e p o r t e d and t h e e l e c t r o n i c r e l a x a t i o n t i m e , x g , f o r s p i n - l a b e l e d lysozyme i s a b o u t 3.5 x 1 0 ~ 7 s e c ' . Hence, i t would be e x p e c t e d t h a t x $ f o r the n i t r o x i d e S L - S e r 195 t r y p s i n w i l l be the same o r d e r o f m a g n i t u d e . An e f f e c t i v e s h o r t e n i n g o f x g due t o s p i n exchange a t -3 h i g h c o n c e n t r a t i o n o f n i t r o x i d e (> 10 M) was n e g l i g i b l e i n t h e p r e s e n t e x p e r i m e n t s i n c e S L - t r y p s i n c o n c e n t r a t i o n was a b o u t 1/10 t h e c o n c e n t r a t i o n used i n r e f 10. Now, s i n c e t h e r o t a t i o n a l c o r r e l a t i o n time f o r S L - T r y p s i n i s about one o r d e r o f magnitude s m a l l e r than t h a t o f the e l e c t r o n i c r e l a x a t i o n t ime o f the n i t r o x i d e on the enzyme i t can be s u r e t h a t the dominant d i p o l a r m o d u l a t i o n p r o c e s s w i l l be t h e r o t a t i o n a l m o t i o n o f t h e e n z y m e - i n h i b i t o r complex, i . e . x < 1.6 x 10 s e c c — 3. J u s t i f i c a t i o n f o r the a s s u m p t i o n o f f a s t exchange (s e e APPENDIX B) A l t h o u g h t h e measurement o f t r a n s v e r s e r e l a x a t i o n t i m e a t d i f f e r e n t t e m p e r a t u r e s i n C h a p t e r 3 shows t h a t a l l the s e v e n i n h i b i t o r s are on the f a s t exchange l i m i t , t h i s can n o t be the c r i t e r i o n f o r f a s t exchange i n t h i s e x p e r i m e n t b e c a u s e t h e l i n e w i d t h o f an s p i n l a b e l e d t r y p s i n - i n h i b i t o r complex i s much g r e a t e r t han the l i n e w i d t h o f a t r y p s i n - i n h i b i t o r complex due t o the e l e c t r o n - n u c l e i d i p o l e i n t e r a c t i o n . With t h i s p r e c a u t i o n i n mind, a c o n t r o l e x p e r i m e n t was c o n d u c t e d a t 40°C, and the r e s u l t s a r e - 138 shown i n t a b l e ( 5 . 2 ) . W i t h o u t e x c e p t i o n , ( = — ) f o r each o f t h e s e v e n *2M , i n h i b i t o r s a t 40°C i s s m a l l e r t h a n the c o r r e s p o n d i n g G?—) a t 1 2M 30°C. However, s i n c e ( j — ) . Q 0 i s n o t t o o much s m a l l e r t h a n •i 2M {z=—)__.0 f o r the m - a r r i s . i d i n e , t h i s i n h i b i t o r may f a l l i n t o t h e *2M J U -i n t e r m e d i a t e r a n g e , i . e . between a " f a s t " exchange and "show" exchange l i m i t . S i n c e no c h e m i c a l s h i f t i s o b s e r v e d o n " b i n d i n g t h i s w i l l be c a s e A i n C h a p t e r I and f 1 EI 2 T 2 A + T -EI EI f o r b r i v i t y , L e t L(U = j- - Y~ '2 '2 *2A F o r the d i f f e r e n t exchange l i m i t s 1 f E I F a s t exchange A(y-) = j — , c *EI T E I K T 2 EI (5.15) i n t e r m e d i a t e A(y-) = M T2M + TM T E I ' T 2 EI 1 M slow exchange A(y-) = — , *2 TM T E I > T 2 EI and e q u a t i o n (5.6) r ^ = c o n s t (Tg^) so f a s t exchange 1/6 r o b s r t ( r i s d e t e r m i n e d ) i n t e r m e d i a t e r o b s = c o n s t ^ T2M + T M ^ 1 / 6 > r t ( 9 i v e n u P P e r l i m i t ) s low exchang r Q b s = c o n s t ( x M ) 1 / 6 = r t ( g i v e n upper l i m i t ) - 139 -w h a t e v e r l i m i t a p p l i e s one can always e x t r a c t some d i s t a n c e i n f o r m a t i o n from t h i s k i n d o f measurement. E. R e s u l t and d i s c u s i o n 1. E s t i m a t i o n o f d i s t a n c e s by• m e a s u r i n g , t h e i n d u c e d n u c l e a r r e l a x a t i o n . The r e s u l t s o f e x p e r i m e n t s u t i l i z i n g t he enhanced n u c l e a r r e s o n a n c e l i n e w i d t h s t o c a l c u l a t e d i s t a n c e s between t h e u n p a i r e d e l e c t r o n o f s p i n l a b e l and t h e m e t h y l group o f each bound i n h i b i t o r s a r e g i v e n i n t a b l e 5.1 and t h e r e s u l t s o f t e m p e r a t u r e c o n t r o l e x p e r i m e n t s a t 40°C a r e g i v e n i n t a b l e 5.2. Examples o f t h e o b s e r v e d s p e c t r u m o f one o f t h e i n h i b i t o r s i n t h e p r e s e n c e and absence o f S L - T r y p s i n a r e g i v e n i n f i g u r e 5.8 and 5.9 r e s p e c t i v e l y . From t h e t h r e e d i m e n s i o n a l s t r u c t u r e a t the a c t i v e s i t e s o f 28 D I P - t r y p s i n ( d i i s o p r o p y l f l u o r o p h o s p h a t e i n h i b i t e d t r y p s i n ) , i t seems t h a t the s p i n l a b e l c o v a l e n t l y bound t o t h e y-oxygen o f S e r - 1 9 5 on t r y p s i n c o u l d have two extreme o r i e n t a t i o n s , one w i t h the p i p e r i d i n y l r i n g p r o j e c t i n g o u t i n t o the s o l v e n t , and t h e o t h e r w i t h t h e r i n g p o i n t e d i n w a r d and e x t e n d i n g i n t o h y d r o p h o b i c s p e c i f i c b i n d i n g p o c k e t . I f t h e n i t r o x y l r i n g was p r o j e c t i n g outward i n t o t h e s o l v e n t , then the e t h y l group o f t h e p hosphate e t h y l e s t e r may p a r t i l l y b l o c k t h e e n t r a n c e o f the s p e c i f i c b i n d i n g p o c k e t . S i n c e t h e D I P - t r y p s i n can s t i l l b i n d B e n z a m i d i n e , ( S t r o u d p r i v a t e communication) and s i n c e t h e i s o p r o p y l group i n t h a t e x p e r i m e n t i s l a r g e r t h a n t h e e t h y l group i n t h i s e x p e r i m e n t , a l s o compound ( 3 - m e t h o x y p r o p y l a m i n e ) i s d e f i n i t e l y s m a l l e r t h a n B e n z a m i d i n e ( s p a c e f i l l i n g models) , - 140 -T a b l e 5.1 E x p e r i m e n t a l p a r a m e t e r s used i n enzyme i n t r a m o l e c u l a r c a l c u l a t i o n s ( a t t e m p e r a t u r e 30° ± 1 ° C ) . a. G i v e n i n h e r t z b. G i v e n as M"1 and d e t e r m i n e d by u.v. s t e a d y - s t a t e k i n e t i c s ( C h a p t e r I I I ) . c. Bound r e l a x a t i o n time i n t h e p r e s e n c e o f t r y p s i n ( C h a p t e r IV) d. f g i s a f r a c t i o n o f i n h i b i t o r bound t o enzyme e. E l e c t r o n - n u c l e a r d i p o l e - d i p o l e c o n t r i b u t i o n t o n u c l e a r r e l a x a t i o n o f i n h i b i t o r p r o t o n s i n p r e s e n c e o f S L - t r y p s i n f . D i s t a n c e f r o m -OCH^ ( o r -CHg) o f i n h i b i t o r t o u n p a i r e d e l e c t r o n o f S L - t r y p s i n T a b l e 5.2 E x p e r i m e n t a l p a r a m e t e r s used i n e s t i m a t i o n o f t h e e l e c t r o n - n u c l e a r d i p o l e - d i p o l e c o n t r i b u t i o n t o i n h i b i t o r p r o t o n n u c l e a r r e l a x a t i o n due t o S L - t r y p s i n a t a d i f f e r e n t t e m p e r a t u r e . a. G i v e n i n h e r t z b. I n h i b i t o r : e n z y m e b i n d i n g c o n s t a n t s , g i v e n as M and d e t e r m i n e d by u.v. k i n e t i c ( C h a p t e r I I I ) . With t h e a s s u m p t i o n K, w i l l a n o t v a r y t o o much i n t h e range o f t e m p e r a t u r e 30°C t o 40°C. c. Bound r e l a x a t i o n t i m e i n t h e p r e s e n c e a t t r y p s i n , w i t h t h e a s s u m p t i o n t h a t t h e t e m p e r a t u r e v a r i a t i o n o f ( l / T g g ) i s s m a l l compared t o t h e v a r i a t i o n o f ( l / T g ^ ) on i n c r e a s i n g t h e t e m p e r a t u r e f r o m 30°C t o 40°C. I n h i b i t o r Name Cone, o f I n h i b i t o r (M/a) Cone, o f Enzyme ( M / J I ) X 1 0 4 Observed NMR L i n e w i d t h 3 -0CH 3, -CH 3 o f i n h i b i t o r O b s e r v e d NMR L i n e w i d t h 3 -CH- o f ( C H § ) 3 C - 0 H b 1 c *2B 3 d ( f B x l 0 J ) 1 e ( T H r(A) '2M 1. A c e t a m i d i n e 0.025 0.025 0 1.26 0.36 0.44 0.38 0.52 29 < 5 - -2. 3-methoxypropyl amine 0.03 0.03 0 1.26 0.44 0.76 0.44 0.5 45.5 12 2.42 323 10.7 3. p-methoxybenzylamine 0.015 0.015 0 1.26 0.48 2.64 0.3 0.4 1640 3 S 8.1 766 9.3 4. p - m e t h o x y p h e n y l - 0.025 0 0.62 0.5 435 56 4.62 869 9.1 g u a n i d i n e 0.025 1.26 1.83 0.4 - - - -5. m-methoxyphenyl- 0.025 0 0.48 0.28 2000 67 5.0 574 9.8 g u a n i d i n e 0.025 1.26 1.56 0.36 - - - -6. p - a n i s i d i n e 0.025 0.025 0 1.26 0.36 1.0 0.32 0.4 59 36 3.0 550 9.8 7. m - a n i s i d i n e 0.025 0.025 0 1.26 0.41 0.92 0.4 0.4 11 68 1.1 1087 8.4 143* 68* 3.9 * 82.2* 11.1* T a b l e 5.1 I n h i b i t o r Name Cone, o f Cone, o f Observed NMR Obser v e d NMR i n h i b i t o r S L - T r y p s i n L i n e w i d t h 9 L i n e w i d t h 3 (Ka) (^-) (f„xl(T) ( x H / m O ( r H o n O (M/4) ( M / £ ) x l 0 4 -OCH3 o f -CH 3 o f 12B b !2M 4 U !2M J U i n h i b i t o r s (CH 3 ) 3 C - 0 H 1. A c e t a m i d i n e 2. 3-methoxypropylamine 0.03 1.57 3. p-methoxybenzylamine 0.015 1.57 4. p-methoxyphenyl- 0.025 1.57 g u a n i d i n e 5. m-methoxyphenyl- 0.025 1.57 g u a n i d i n e 6. p - a n i s i d i n e 0.025 1.57 7. m - a n i s i d i n e 0.025 1.57 0.66 2.04 1.92 1.52 1.0 0.84 0.46 0.3 0.4 0.32 0.4 0.36 45.5 12 3.02 1640 36 10.00 435 56 2000 67 59 36 11 68 5.75 6.15 3.74 196 423 765 444 434 323 766 869 574 550 1.32 1050 1087 T a b l e 5.2 - 143 -F i g u r e 5.8 T y p i c a l P r o t o n H i g h - R e s o l u t i o n Spectrum o f an I n h i b i t o r o f T r y p s i n P r o t o n NMR s p e c t r u m o f p - m e t h o x y p h e n y l g u a n i d i n e (0.025 M) i n 0.1 M B o r a t e b u f f e r , Ph 7.55, 0.02 M C a C l g , s p e c t r a l w i d t h 400 Hz; A c q u i s i t i o n t i m e = 5 s e c . 10 p o i n t s / H e r t z . Spectrum r e p r e s e n t s the F o u r i e r t r a n s f o r m o f t h e sum o f 4 t r a n s i e n t s ; f i e l d - f r e q u e n c y l o c k based on DgO. [no t r y p s i n p r e s e n t ] I n s e r t : The Methyl group o f t h e i n h i b i t o r and t h e i n t e r n a l s t a n d a r d ( t e r t i a r y b u t y l a l c o h o l ) a r e shown on a 20X - expanded h o r i z o n t a l s c a l e . i - 145 -F i g u r e 5.9 T y p i c a l E f f e c t o f SL - T r y p s i n on P r o t o n NMR Spectrum o f an I n h i b i t o r P r o t o n NMR s p e c t r u m o f p - m e t h o x y p h e n y l g u a n i d i n e (0.025 M) i n x l O " 3 t h e p r e s e n c e o f 1.26 M SL TRYPSIN. The b u f f e r i s t h e same as i n F i g u r e 5.8; s p e c t r a l w i d t h i s 400 Hz; a q u i s i t i o n t i m e 5 s e c , 10 p o i n t s / H e r t z ; 25 t r a n s i e n t s , DgO l o c k . I n s e r t : The h o r i z o n t a l s c a l e f o r t h e s p e c t r u m o f t h e methyl groups o f t h e i n h i b i t o r and o f t h e i n t e r n a l s t a n d a r d ( t e r t i a r y b u t y l a l c o h o l ) a r e expanded 10 and 20 t i m e s , r e s p e c t i v e l y . - 147 -compound 2 s h o u l d have no d i f f i c u l t y i n f i n d i n g i t s way i n t o the s p e c i f i c b i n d i n g p o c k e t . Now compound 3 (p-methoxybenzylamine) i s a l s o a c o m p e t i t i v e i n h i b i t o r (shown by U.V. i n C h a p t e r I I I ) , and 0 i s a bout 2A l o n g e r than b e n z a m i d i n e , so whether 3. can b i n d t o the s p e c i f i c b i n d i n g p o c k e t o r n o t w i l l depend on the d i s p o s i t i o n o f the end methyl group o f the s p i n l a b e l r e l a t i v e t o methoxy group a t the p a r a p o s i t i o n o f Compound 3. A l t h o u g h some s t e r i c h i n d r a n c e may be p r e s e n t , b u t s t i l l b i n d i n g o f 3 t o t h e s p e c i f i c p o c k e t can n o t be r u l e d o u t c o m p l e t e l y . Due t o t h e i r l o n g e r l e n g t h and b e n t shape, compounds 4 and 5 (P - and m-methoxylphenyl g u a n i d i n e . H c l ) a r e u n l i k e l y be bound i n the s p e c i f i c b i n d i n g p o c k e t . Compounds 6 and 7 (p- and m - a n i s i d i n e ) a r e n e u t r a l , and thus t h e i r chance t o be bound i n s p e c i f i c p o c k e t w i l l be s l i m . Suppose the p i p e r i d i n y l group i s p o i n t e d towards the s p e c i f i c b i n d i n g p o c k e t and s i t s s n u g l y i n the p o c k e t . Then none o f the i n h i b i t o r s s t u d i e d h e r e w i l l be a b l e t o b i n d i n the s p e c i f i c b i n d i n g p o c k e t . Hence t h e o b s e r v e d l i n e b r o a d e n i n g o f compound 2 and 3 c o u l d o n l y be e x p l a i n e d by s u p p o s i n g t h a t b o t h o f them can a l s o b i n d t o a s e c o n d a r y s i t e . T h i s i s p o s s i b l e b e c a u s e compounds 2 and 3 a r e n o t t h a t d i f f e r e n t i n s i z e from compounds 4 and 5 ; i f t h e s e c o n d a r y s i t e s a r e i n an exposed r e g i o n o f the enzyme s u r f a c e , then the r e q u i r e m e n t o f s t e r i c s p e c i f i c i t y w i l l n o t be so s e v e r e as t h a t o f the s p e c i f i c b i n d i n g p o c k e t . The r e a s o n why compounds 2 and 3 d i d n o t show any s i m i l i a r b e h a v i o r i n the U.V. s t u d i e s ( C h a p t e r I I I ) w o u l d then be t h a t compounds 2 and 3 must be bound a t the s e c o n d a r y s i t e i n such a way t h a t t h e a c c e s s o f s u b s t r a t e - 148 -(D.L. BAPA) t o the s p e c i f i c b i n d i n g p o c k e t i s p r e v e n t e d . Whereas the b i n d i n g o f compounds 4.5.6 and 7 a t the s e c o n d a r y s i t e a c t s t o f a c i l i t a t e t h e b i n d i n g o f D.L-BAPA ( C h a p t e r I I I ) , p e r h a p s t h i s was due t o the e x t r a range o f h y d r o p h o b i c e n v i r o m e n t f u r n i s h e d by t h e s e bound i n h i b i t o r s . From t h e U.V. r e s u l t s , t h e d i s t a n c e s measured f o r t h e i n h i b i t o r s m i g h t be e x p e c t e d t o be c l a s s i f i e d i n t o t h r e e k i n d s , c o m p e t i t i v e , n o n c o m p e t i t i v e ( c h a r g e d and n e u t r a l ) . The s u r p r i s i n g l y s i m i l a r o i n h i b i t o r - s p i n l a b e l d i s t a n c e s ( a l l around 10A) f o u n d h e r e h i g h l y s u g g e s t t h a t t h e r e i s o n l y one s e c o n d a r y b i n d i n g s i t e . S i n c e t h e E.S.R. s p e c t r u m o f t h e S.L. T r y p s i n d i d n o t change i n t h e PH range o f 3 t o 8 w h i l e a t the same t i m e t h e a c t i v i t y o f t r y p s i n v a r i e s by many o r d e r s o f magnitude,no m a j o r c o m f o r -m a t i o n a l change a t the a c t i v e s i t e seems l i k e l y . The s u b s t r a t e a c t i v a t i o n ^ and t h e f a c i l i t a t e d b i n d i n g b e h a v i o r shown by some o f the i n h i b i t o r s ( C h a p t e r I I I ) can a l l be e x p l a i n e d by h a v i n g s e c o n d a r y b i n d i n g s i t e c l o s e t o t h e s p e c i f i c b i n d i n g p o c k e t . E x t r a e v i d e n c e t o s u p p o r t t h i s model i s t h a t t h i o n i n e ^ w i t h a l a r g e s i z e , i s a l s o a c o m p e t i t i v e i n h i b i t o r . I t may w e l l be bound t o the s e c o n d a r y s i t e and a t t h e same t i m e a c t t o b l o c k the e n t r a n c e o f the p r i m a r y s i t e , thus showing a p p a r e n t c o m p e t i t i o n i n h i b i t i o n b e h a v i o r . F u r t h e r d i s t a n c e i n f o r m a t i o n can be f o u n d i n t a b l e 5.3. The sum o f t h e l e n g t h o f the i n h i b i t o r (on the l o n g e r a x i s ) and t h e o d i s t a n c e s e s t i m a t e d h e r e a r e a l l g i v e n around 20.5 ± 0.4 A f o r o the c h a r g e d i n h i b i t o r s , and 19.1 ± 1.1 A f o r the n e u t r a l i n h i b i t o r s . -149-T a b l e 5.3 L e n gth o f i n h i b i t o r m o l e c u l e s and the p o s s i b l e l o c a t i o n o f s e c o n d a r y b i n d i n g s i t e s on t r y p s i n . Shown i n t h e T a b l e a r e : the m o l e c u l a r l e n g t h o f each i n h i b i t o r ( L ) ; t h e d i s t a n c e ( r ) between t h e s p i n - l a b e l u n p a i r e d e l e c t r o n and t h e i n h i b i t o r - C ^ o r -OCH^ g r o u p , e s t i m a t e d from s p i n l a b e l - i n d u c e d n u c l e a r r e l a x a t i o n ; and t h e sum o f t h e s e two terms. See t e x t f o r d i s c u s s i o n . Name acetamidine 3-methoxypropy1 ami ne p-methoxybenzy1 amine p-methoxyphenylguani di ne m-methoxypheny1guani di ne p - a n i s 1 d i n e m-anisidine Structure H H L(A) r ( A ) L + r ( A ) 5.8 9.5 10.7 20.2 11.4 9.3 20.7 11.8 9.1 20.9 10.8 9.8 20.6 10.4 9.8 20.2 9.6 8.4 18.0 11.1* 2 0 . 7 * - 151 -F o r l o n g e r i n h i b i t o r s , the d i s t a n c e between the methyl group o f the i n h i b i t o r and the u n p a i r e d e l e c t r o n o f the s p i n l a b e l i s s h o r t e r , w h i l e the c o n v e r s e s i t u a t i o n a p p l i e s t o the s h o r t e r o n e s . I t seems h i g h l y u n l i k e l y t h a t t h e r e c o u l d be more than one s e c o n d a r y s i t e a t around 10A f r o m the s p i n l a b e l . From the t w o - p u b l i s h e d c o l o r s t e r e o diagrams on D I P - t r y p s i n , i t i s r a t h e r d i f f i c u l t t o s p e c u l a t e as t o the l o c a t i o n the s e c o n d a r y b i n d i n g s i t e . With the r e a l t h r e e d i m e n s i o n a l s t r u c t u r e , one s h o u l d be a b l e t o narrow the p o s s i b i l i t i e s and l o c a t e t h e s e c o n d a r y s i t e . The d i s c r e p a n c y o f the b i n d i n g c o n s t a n t s ( d i s c u s e d i n C h a p t e r 4) measured by U.V. and by NMR s h o u l d n o t pose t o o much t r o u b l e . Even i f the b i n d i n g c o n s t a n t d i f f e r e n c e between t h e s e two methods were abo u t one o r d e r o f m agnitude, t h e NMR e s t i m a t e d d i s t a n c e ( r ) would o o n l y change by a b o u t 2A, F o r example, f o r m - a n i s i d i n e , i f one uses the b i n d i n g c o n s t a n t o b t a i n e d from NMR c o n c e n t r a t i o n d a t a , t h e o d i s t a n c e t u r n s o u t t o be 11.1A, thus b r i n g the o n l y anomalous r e s u l t back i n t o the p a t t e r n : i . e . , the sum o f t h e l e n g t h o f t h i s i n h i b i t o r and the e s t i m a t e s d i s t a n c e s f o r a l l the i n h i b i t o r s w i l l o now come o u t t o be a b o u t 20.5 ± 4A, 2. C o n f o r m a t i o n a l changes examined by ESR From the c o n t r o l e x p e r i m e n t , the s e c t i o n on p r e p a r a t i o n o f S L - t r y s i n , an a c t i v e t r y s i n - f r e e , s p i n - l a b e l e d t r y s i n has been p r e p a r e d ( f i g u r e 5.5). T h i s S L - t r y p s i n w i l l be h y d r o l i z e d by a d d i n g commercial t r y p s i n ; ( F i g 5.6) a l s o some o f i t w i l l be d e n a t u r e d d u r i n g the p r o c e s s o f l y o p h o l i z a t i o n ( F i g . 5.4). But the - 152 -d e n a t u r e d S L - t r y p s i n w i l l n o t c o n s t i t u t e more than a few p e r c e n t ^ o f the t o t a l p r o t e i n . The ESR s p e c t r a o f S L - t r y p s i n , a f t e r i n c u b a t e s 30 - 45 m i n u t e s i n the NMR probe a t 40°C, was n o t changed as shown by c o m p a r i s o n t o the ESR s p e c t r a o f the l y o p h o l i z e d S L - t r y p s i n ( F i g u r e 5.4). T h i s i s a n o t h e r good i n d i c a t i o n o f l a c k o f a u t o p r o t e o l y s i s . The f a c t t h a t C a + + can enhance t h e e f f i c i e n c y o f the c a t a l y s i s o f t r y p s i n towards c e r t a i n s u b s t r a t e s has been s u g g e s t e d t o a r i s e from c o m f o r m a t i o n a l changes a t t h e a c t i v e s i t e 3 ! . j n u s ^ comparing t h e ESR s p e c t r u m o f the l a b e l e d t r y p s i n i n the p r e s e n c e and t h e absence o f C a + + o r M u + + , one m i g h t hope t o d e t e c t such d i v a l e n t metal i o n i n d u c e d c o n f o r m a t i o n a l changes. A l s o t h e s u s t r a t e a c t i v a t i o n r e p o r t e d by L a s K o w s k i 1 1 and the i n t e r a c t i o n among the i n h i b i t o r s o b s e r v e d by o u r U.V. s t e a d y s t a t o K i n e t i c s t u d i e s i n C h a p t e r 3. A l l s u g g e s t the e x i s t a n c e o f a s e c o n d a r y b i n d i n g s i t e . T h i s was p r o v e d by t h e NMR l i n e w i d t h s t u d i e s i n t h i s c h a p t e r . The d i s t a n c e s between the n i t r o x i d e f r e e r a d i c a l and t h e i n h i b i t o r s bound t o t h e s e c o n d a r y s i t e was e s t i m a t e d o t o be a r o u n d 10A. Now i f any changes had a p p e a r e d i n t h e E.S.R s p e c t r u m o f the s p i n - l a b l e d engyme upon a d d i n g the i n h i b i t o r o r s u b s t r a t e , one would have a d d i t i o n a l i n s i g h t i n t o the e f f e c t o f i n h i b i t o r bound a t the s e c o n d a r y s i t e on the c o n f o r m a t i o n on a t the a c t i v e s i t e . U n f o r t u n a t e l y a c a r e f u l e x a m i n a t i o n o f the s p e c t r a ( F i g u r e 5.7) showed no d e t e c t a b l e changes upon any o f the a b o v e - l i s t e d p u r t u b a t i o n s . The o n l y c o n c l u s i o n t o be drawn here i s t h a t the s p i n l a b e l changes o r ESR l i n e shape i s weak i n t e r a c t i o n s - 153 -i n s e n s i t i v e t o the s m a l l w i t h bound i n h i b i t o r s . c o m f o r m a t i o n a l F. Summary The a c t i v e t r y p s i n - f r e e , S.L. t r y p s i n was p r e p a r e d s u c e s s f u l l y . The f a s t exchange l i m i t f o r the s p i n l a b e l e x p e r i m e n t was re-examined and d i s c u s s e d . Through the i n d u c e d l i n e b r o a d i n i n g o f the Methyl group o f the bound i n h i b i t o r s , t h e d i s t a n c e between the methyl group o f the i n h i b i t o r s a t the s e c o n d a r y s i t e and the u n p a i r e d e l e c t r o n o o f the s p i n - l a b e l a t s e r i n e 195 was e s t i m a t e d t o be around 10A. I t ' s i m p l i c a t i o n s was d i s c u s s e d w i t h the h e l p o f 3 d i m e n s i o n a l s t r u c t u r e o f DIP- and B A - t r y p s i n . E.S.R s p e c t r u m o f the S.L. t r y p s i n w i t h d i f f e r e n t p u r t u b a t i o n s was s t u d i e d , and found t h a t m i n o r c o m f o r m a t i o n a l changes w i l l n o t p r o d u c e an o b s e r v i a b l e change i n the ESR s p e c t r u m . - 154 -R e f e r e n c e s 1. S. O h n i s h i , and H.M. M c C o n n e l l , J . Amer. Chem. Soc. 87, 7293 ( 1 9 6 5 ) . 2. W.L. H u b b e l l and H.M. M c C o n n e l l , P r o c . Nat. Acad. S c i , U.S.A. 61_., 12 ( 1 9 6 8 ) . P r o c . Nat. Acad. S c i . 63, 16, (1969 b ) . 3. H.M. M c C o n n e l l , S. Ogawa and A. H o r w i t z . N a t u r e , Lond. 720, 787 ( 1 9 6 8 ) . 4. H.M. Mc C o n n e l l and B.G. M c F a r l a n d , Q u a r t . Rev. B i o o h y s . 3_, 91 ( 1 9 7 0 ) . 5. I . C P . S m i t h , B i o l o g i c a l A p p l i c a t i o n o f e l e c t r o n s p i n r e s o n a n c e s p e c t r o s c o p y , W i l e y i n t e r s c i e n c e N.Y. 6. A.S. M i l d v a n and H. W e i n e r , J . B i o l . Chem. 244, 2465 ( 1 9 6 9 ) . 7. M. Cohn and J . Reubeu, A c c o u n t s Chem. Res. 4, 6 ( 1 9 7 1 ) . 8. A. B e n n i c k . , I.D. Ca m p b e l l , R.A. Dwek, N . C P r i c e , G.K. Radda, and A.G. Salmon, N a t u r e ( L o n d o n ) , New B i o l 234, 140. 9. R.D. K o r n b e r g , H.M. M c C o n n e l l , B i o c h e m i s t r y TO, 1111 ( 1 9 7 1 ) . 10. R.W. Wien, J.D. M o r r i s e t t , and H.M. M c C o n n e l l , 3707 ( 1 9 7 2 ) . 11. M. L a z d u n s k i and M. D e l a a g e , B i o c h i m . B i o p h y s . A c t a 105, 541 ( 1 9 6 5 ) . 12. T. Inagami and S.S. Y o r k , Biochem. ]_, 4045 (1968) 13. B.M. Sanborn and G.E. H e i n , Biochem. 7_, 3616 (1968) 14. J.D. M o r r i s e t t and C A . B r o o m f i e l d , j . o f B i o l . Chem, 247 7224 ( 1 9 7 2 ) . 15. J.D. M o r r i s e t t , C A . B r o o m f i e l d , and 3.E. H a c k l e y , JR. 244, 5758 ( 1 9 6 9 ) . 16. C . J . B e r l i n e r and S.S. Wong, J . B i o l . Chem. 248, 1118 ( 1 9 7 3 ) . 17. M.A. C o l e t t i - P r e v i e r o , A. P r e v i e r o and E. Z u c k e r k a n d l , J . Mol. B i o l . 39, 493 ( 1 9 7 3 ) . 18. Sephadex Gel F i l t r a t i o n i n t h e o r y and p r a c t i c e , p u b l i s h e d by pharmaeia f i n e c h e m i c a l s , Sweden. - 155 -19. M. L a s k o w s k i . J r . and R.W. S e a l o c k , The Enzymes. Vol 3. p.403. t h i r d E d i t i o n (Ed by P.D. Boyer) 1971. 20. D.D. S h r o e d e r and E. Show, J . B i o l . Chem. 243, 2943 ( 1 9 6 8 ) , 21. M. K u n i t z , J . Gen. p h y s i o l . 30 , 291 ( 1 9 4 7 ) . 22. C. T. T r o w b r i d g e , A. K r e h b i e l , M. L a s k o w s k i , JR., Biochem 2, 843 ( 1 9 6 3 ) . 23. P.K. G l a s o e , F.A. Long, J . phys. Chem. 64, 188 ( 1 9 6 0 ) . 24. R.P. Ha n g l a n d and L. S t r y e r , Comformation o f b i o p o l y m e r s ( e d i t e d by G.N. Ramachandran). Vol 1, 1967, Academic P r e s s , N.Y, 25. H.R. M a h l e r and E.H. Cor d e s , B i o l o g i c a l C h e m i s t r y H a r p e r and Row. p. 275, t a b l e 6.6 ( 1 9 6 6 ) . 26. R.G. K o o s e r , V.W. V a l l a r d and J.H. F r e e d , J . Chem. phys. 50, 5243 ( 1 9 6 9 ) . ~~ 27. R.W. Wien, J.D. M o r r i s e t t , H.M. M c C o n n e l l , B i o c h e m i s t r y 11, 3707 ( 1 9 7 2 ) . ~~ 28. R.M. S t r o n d , L.M. Kay and R.E. D i c k e r s o n , c o l d s p r i n g H a r b o r Sym. Quant. B i o l . 36, 125 ( 1 9 7 1 ) . 29. R.M. S t r o n d , ( S p e c i f i c b i n d i n g o f t r y p s i n ) , p r i v a t e Communication, 30. A.N. G l a z e r , J . B i o l . Chem. 242, 3326 ( 1 9 6 7 ) . 31. N.M. Green and H. N e u r a t h , J . B i o l . Chem. 204, 379 ( 1 9 5 3 ) , - 156 -Appendix A. The L o n g i t u d i n a l R e l a x a t i o n Time T, f o r A Three S i t e System. The l o n g i t u d i n a l r e l a x a t i o n time T, f o r a t h r e e s i t e system can be d e r i v e d as f o l l o w s ( A . l ) F o r t h i s s y s t e m , M c C o n n e l l ' s e q u a t i o n s may be w r i t t e n . A + M B + E I* 1* B A + ME ^ C where M i s metal i o n and E i s enzyme, and ME i s m e t a l -enzyme complex, (A.2) d M . d t T A ( k , [ M ] + k 3 [ M E ] ) M zA + k ^ M ^ + k_ 3M dM. d t dM r M z B - M 0 B ^ J + K ^ M J M / - ( k _ 1 + k 2 [ E ] ) M z ° + k _ 2 B , M C z (A.3) •z " M o "dt 1 T ^ ) + k 3 [ M E ] M z " + k 2 [ E ] M z B - ( k , 2 + k _ 3 ) M 2 - 157 -From the c o n c e n t r a t i o n c o n d i t i o n s t h a t [A] » [B] o r [C] i t f o l l o w s t h a t M z » M 8 o r M z . Then dM 8 dM C - a r = - a l • 0 < A - 4 > F o r b r e v i t y , l e t M A = A , M B = B . M ° = C , M A = A . M 8 = B . M ° = C z z z z z z o o o o o o two simutaneous e q u a t i o n s r e s u l t : - ( Z B °) - ( k ^ + k 2 [ E ] ) B z + k 1 [ M ] A z + k _ 2 C z = 0 (A.5) T t '1 c - c - ( Z c °) - ( k _ 2 + k _ 3 ) C z + k 3 [ M E ] A z + k 2 [ E ] B z = 0 (A.6) T l B z and C z can then be s o l v e d i n terms o f A z and s u b s t i t u t e d i n t o the f i r s t e q u a t i o n o f ( A . 3 ) . A f i r s t o r d e r d i f f e r e n t i a l e q u a t i o n c a n then be obtained,, dAj: d t [ 1 k , M M ] | ~ T ~ k l [ M ] _ K 3 [ M E ] + ~T ( ~ T ~ + K - 2 K 3 [ M E ] ^ ' T-j C k _ 3 / T i i k ? M M E ] ) -2 B C 1 1 T B I 1 A o k - l f f' k ? ' T l T C T 1 T l + 3 ( — i K - 2 TgTpFT^ T , 8 T, f f ' k ? f + - r > K (A.7) where - 158 -i - = k , + k 2 [ E ] ) 1 B n = f A . C = f'A 0 0 0 0 and F = YT~ ~ k - 2 k 2 t E ] * ( A - 8 > F o r t h e f i r s t o r d e r d i f f e r e n t i a l e q u a t i o n dA " d f = - P A Z + Q (A . 9 ) the s o l u t i o n i s A z = ( c o n s t ) e p t + S. ( A J O ) S i n c e i s t h e time c o n s t a n t f o r the r e l a x a t i o n a l o n g t h e z a x i s , A z = ( c o n s t ) e ~ t / T l + £ (A.11) and 1 p - - yj-e q u a t i o n s (A.12) can then be o b t a i n e d : 2 • i 1 r r- T \ / 1 1 1 T C + k-2 + k-3 - 159 -k - i + T r f ( 7 B + k . i + k _ 2 [ E ] ) -3 ^ [ M ] 1- '-2 1 ( + k - 1 + k 2 [ E ] ) ( - L r + k . 2 + k _ 3 ) - k . 2 k 2 'l 'l [ E ] [ k - i + r | ( ^ + k - i + ¥ E ] ) ] k 3 [ M E ] , (A.12) T h i s g e n e r a l s o l u t i o n f o r t h r e e s i t e s y s t e m can be r e d u c e d t o t h r e e s i m p l e c a s e s . Case 1. o n l y A i s p r e s e n t r = " 7 ( A - 1 3 ) Case 2. o n l y A and B ( o r A and C) a r e p r e s e n t 9 t h e n r = - r + — ? B ( A J 4 ) 1 T t 1 + k - i T i w i t h Thus k - i • \ - ¥ M ' - w - f B ' T l T l + T B - 160 -Case 3. t h e exchange r a t e s between B and C a r e e x t r e m e l y s l o w 3 i .e. kg = k _ 2 = 0 T = - \ + ( \ ) ki [M] + ( 1 ? ) k, [ME] (A.16) With (A.16) and ^ . J C L . f ( ; . then r e s u l t t h e e q u a t i o n (2.7) - 161 -A p p e n d i x B. Comments on f a s t exchange l i m i t f o r s p i n l a b e l enhancement o f n u c l e a r r e l a x a t i o n . The i n e q u a l i t y (7) employed i n Wien's work^ on s p i n - l a b e l e d l ysozyme f o r t h e c o n d i t i o n o f f a s t exchange i s n o t g e n e r a l l y t r u e : f f T T ' T T '2A 12M A l g e b r a i c a l l y , one can n o t g e t t o e q u a t i o n (9) o f r e f . 1 by u t i l i z i n g t h e two i n e q u a l i t i e s (7) and (8) i n t h a t p a p e r . In o r d e r t o o b t a i n t h e e q u a t i o n (9) i n t h e i r w ork s the 2 n e c e s s a r y and s u f f i c i e n t c o n d i t i o n i s T M T A 1 Y1-, » 1 and — » Aw m 1 2 M 1 2 A T M m f o r t h e f i r s t exchange l i m i t t o be t r u e , 3 A c o n t r o l e x p e r i m e n t a t d i f f e r e n t t e m p e r a t u r e f o r each o f th e i n h i b i t o r s s t u d i e d as we d i d i n c h a p t e r 4 s h o u l d be a b l e t o r e s o l v e which p r o c e s s dominates t h e NMR l i n e b r o a d e n i n g , Even i n some f a v o r a b l e c a s e s , i n e q u a l i t i e s (7) and (8) o f r e f . 1 c o u l d l e a d t o a f a s t exchange l i m i t . But f o r the i n h i b i t o r s NAG, di-NAG o f lysozyme t he t e m p e r a t u r e s t u d i e d 4 showed t h a t the c h e m i c a l exchange dominated t he t r a n s v e r s e r e l a x a t i o n t ime i . e . i n the slow exchange l i m i t 1 1 1 1 T » T >> » T~ - 162 -0 r T A > T2A> TM > T2M" With s p i n - l a b e l e d l y s o z y m e , t h e terms =-!—, Y~- can o n l y 12A 12M be made l a r g e r by the d i p o l a r i n t e r a c t i o n and c o n t a c t i n t e r a c t i o n i n the p r e s e n c e o f f r e e r a d i c a l s i n the s o l u t i o n . I f one assumes as t h o s e a u t h o r s d i d , t h a t the a s s o c i a t i o n c o n s t a n t (as w e l l as o f f r a t e o f each i n h i b i t o r 1 ) a r e the same f o r s p i n - l a b e l e d l y s i z y m e and o r d i n a r y l y s o z y m e , then f a s t exchange l i m i t can n o t r e s u l t f r o m an i n c r e a s e d ( = — ) i n g o i n g !2M f r o m o r d i n a r y lysozyme t o s p i n - l a b e l e d l y s o z y m e . Thus t h e l i n e b r o a d e n i n g f o r S L - l y s o z y m e i n r e f . 1 i s due m a i n l y t o t h e p r o c e s s o f c h e m i c a l exchange, r a t h e r t h a n the "bound" Tg, i n c o n t r a s t t o t h e c l a i m o f r e f . 1 . F o r slow exchange, x^ » T g ^ , t h e d i s t a n c e e s t i m a t e d f r o m t h e l i n e b r o a d e n i n g s h o u l d p r o v i d e a c r u d e h i g h e r l i m i t t o t h e c o r r e s p o n d i n g d i s t a n c e r a t h e r t h a n t h e c r u d e l o w e r l i m i t as the a u t h o r c l a i m e d . REFERENCES 1. R.W. w i e n , J.D. M o r r i s e t t , and H.M. M c C o n n e l l , ]_1_,3707 (1972) 2. J.A. P o p l e , W.G. S c h n e i d e r and H.J. B e r n s t e i n , High R e s o l u t i o n NMR, P. 221, M c G r a w - H i l l Book Company (1959) 3. B.D. S y k e s , P.G. S c h m i d t and G.R. S t a r k , J . B i o l . Chem.,245, 1180,(1970) 4. B.D. Sykes and C. P a r r a v a n o , J . B i o l . Chem. 244,3900 (1969)

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Magnetic resonance studies of trypsin Kang, Shyue-yue 1974

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Magnetic resonance studies of trypsin Kang, Shyue-yue 1974

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