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UBC Theses and Dissertations

Magnetic resonance studies of trypsin Kang, Shyue-yue 1974

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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. 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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 . 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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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