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UBC Theses and Dissertations

Nuclear magnetic resonance studies on interactions of active site inhibitors with acetylcholinesterase Carruthers, Junko Maetani 1980

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NUCLEAR MAGNETIC RESONANCE STUDIES ON INTERACTIONS OF ACTIVE SITE INHIBITORS WITH ACETYLCHOLINESTERASE by UNKO MAETANI CARRUTHERS B.Sc, Simon Fraser University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1980 (g) Junko Maetani Carruthers, 1980 I n 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 t h e 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 a t 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 n o t 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 . D e p a r t m e n t o f rJMb/wuV^ 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 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 n„tP <kjJr S i hyp ABSTRACT A c e t y l c h o l i n e s t e r a s e i s an enzyme which p l a y s an i m p o r t a n t p a r t i n n e u r a l t r a n s m i s s i o n . T h i s enzyme, however, has not been e x t e n s i v e l y i n v e s t i g a t e d , e s p e c i a l l y a t the m o l e c u l a r l e v e l , due t o the d i f f i c u l t i e s i n o b t a i n i n g pure and w e l l c h a r -a c t e r i z e d forms of the enzyme. T h i s t h e s i s d e s c r i b e s a method f o r p r e p a r i n g such a form of AchE from E l e c t r o p h o r u s e l e c t r i c u s , f o l l o w e d by NMR s t u d i e s on the i n t e r a c t i o n between t h i s form and v a r i o u s i n h i b i t o r s . I n h i b i t o r s range i n s i z e from a s m a l l trimethylammonium i o n t o a l a r g e a t r o p i n e m o l e c u l e , but a l l c o n t a i n a q u a t e r n a r y ammonium group. In c o n t r a s t t o p r e v i o u s -l y r e p o r t e d l a r g e l i n e b r o a d e n i n g s of i n h i b i t o r NMR resonances upon i n t e r a c t i o n w i t h n a t u r a l AchE, the p r e s e n t r e s u l t s showed no s i g n i f i c a n t l i n e b r o a d e n i n g w i t h any e x c e p t one of the i n -h i b i t o r s i n the same c o n c e n t r a t i o n range. In o r d e r t o f u r t h e r i n v e s t i g a t e the b i n d i n g s i t e i n the a c t i v e c e n t e r of the en-zyme, the a c t i v e e s t e r a t i c s i t e was permanently b l o c k e d by s u l f o n y l a t i o n . I n h i b i t o r s i n t e r a c t i n g w i t h t h i s m o d i f i e d AchE indeed show l i n e b r o a d e n i n g s , g i v i n g r i s e t o bound l i n e w i d t h s which are w e l l w i t h i n the e x p e c t e d t h e o r e t i c a l l i m i t . The cause of the d i f f e r e n c e s i n observed l i n e w i d t h s of i n h i b i t o r s between n a t u r a l and m o d i f i e d AchE i s d i s c u s s e d i n terms of changes i n exchange r a t e s and f l e x i b i l i t y of bound i n h i b i t o r s . The p r e s e n t o v e r a l l r e s u l t s c o n f i r m the importance of p r i o r c h a r a c t e r i z a t i o n o f the enzyme hence e x p l a i n i n g the d i s c r e p a n c y between the l i n e b r o a d e n i n g s of i n h i b i t o r s i n t e r a c t i n g w i t h n a t u r a l AchE observed by another worker and ones observed i n c u r r e n t work, and a l s o i n d i c a t e t h a t the a c t i v e r e g i o n of n a t u r -a l AchE i s l i k e l y t o be l a r g e , the e f f e c t i v e s i z e b e i n g a t l e a s t the s i z e of a t r o p i n e , and/or l o c a t e d on the s u r f a c e of the en-zyme . - iv -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES AND TABLES v i i ACKNOWLEDGEMENT ix CHAPTER I: INTRODUCTION 1 A. Acetylcholinesterase 1 B. Extraction and P u r i f i c a t i o n 4 C. Structure 6 i) The 9S, 14S and 18S Species 7 i i ) The 11S Species 11 D. C a t a l y t i c Mechanism 12 i) Substrate Reaction 13 i i ) Inhibitors and Their Interaction with the Enzyme 15 E. Spectroscopic Techniques in the Study of the Cationic Mechanism of Acetylcholinesterase 20 CHAPTER II : THEORETICAL BACKGROUND 25 A. The Lineshape Analysis for a Molecule Undergoing Chemical Exchange when the Population of the Two Sites are Greatly Different 25 B. Expected Linewidth in NMR Spectra for a Methyl Group Attached to a Macromolecule 32 C. Plots to Obtain Bound Linewidth 34 CHAPTER I I I : EXPERIMENTAL METHODS A. P u r i f i c a t i o n of Acetylcholinesterase 36 i) Extraction of Acetylcholinesterase from E l e c t r i c Eel in High Salt Media 36 - V -Page i i ) P u r i f i c a t i o n o f A c e t y l c h o l i n e s t e r a s e by A f f i n i t y Chromatography 37 a) P r e p a r a t i o n of the Column 37 b) Running of the Column 39 i i i ) A c e t y l c h o l i n e s t e r a s e Assay 41 B. C o n v e r s i o n of A c e t y l c h o l i n e s t e r a s e t o 11S Form .. 42 i ) T r y p s i n D i g e s t i o n 42 i i ) C h a r a c t e r i z a t i o n of Converted A c e t y l -c h o l i n e s t e r a s e 43 C. P r e p a r a t i o n of A c e t y l c h o l i n e s t e r a s e Samples i n D 20 B u f f e r 46 i ) C o n c e n t r a t i o n o f the Enzyme w i t h the Amicon F i l t e r 46 i i ) Other methods f o r C o n c e n t r a t i n g AchE i n t o D 20 B u f f e r 47 D. P r e p a r a t i o n of M o d i f i e d Enzyme and i t s V e r i f i c a t i o n 48 i ) P r e p a r a t i o n o f E s e r o l i n e 48 i i ) S u l f o n y l a t i o n of A c e t y l c h o l i n e s t e r a s e 50 E. S t a b i l i t y o f the Enzyme 51 F. NMR Measurements 52 CHAPTER IV: RESULTS 5 4 A. S t a b i l i t y and P u r i t y o f the Enzyme 54 B. I n h i b i t o r s w i t h U n m o d i f i e d Enzyme 56 C. E s e r i n e w i t h Carbamylated Enzyme 64 D. I n h i b i t o r s w i t h M e t h a n e s u l f o n y l a t e d A c e t y l -c h o l i n e s t e r a s e 71 CHAPTER V: DISCUSSION 79 A. I n h i b i t o r s w i t h U nmodified AchE 79 - v i -Page B. Inhibitors with Modified Enzyme 83 C. Possible Extention of Studies 88 GLOSSARY 94 REFERENCES 95 - v i i -LIST OF FIGURES AND TABLES F i g u r e Page I - l Model o f the 18S A c e t y l c h o l i n e s t e r a s e 8 1-2 M u l t i p l e Forms o f A c e t y l c h o l i n e s t e r a s e from E. E l e c t r i c u s 9 1-3 The R e a c t i o n Squeme o f the H y d r o l y s i s o f A c e t y l c h o l i n e by A c e t y l c h o l i n e s t e r a s e 14 1-4 B i n d i n g S i t e s o f A c e t y l c h o l i n e s t e r a s e 16 1-5 S t r u c t u r e o f AchE I n h i b i t o r s 18 I I - l A P l o t o f 1/T 2 v s . R e c i p r o c a l R o t a t i o n a l C o r r e l a t i o n Time : 33 I I I - l An E l u t i o n P r o f i l e of A c e t y l c h o l i n e s t e r a s e from an A f f i n i t y Column 40 I I I - 2 S u c r o s e G r a d i e n t P r o f i l e o f A c e t y l c h o l i n e s t e r a s e b e f o r e and a f t e r the Treatment w i t h T r y p s i n 45 I I I - 3 An Enzyme D i s t r i b u t i o n i n a Tube a f t e r C e n t r i f u g a t i o n 49 IV-1 Sucrose G r a d i e n t P r o f i l e of 11S AchE b e f o r e and a f t e r the S t o r a g e i n 4M NaCl B u f f e r a t 4°C 55 IV-2 Sucrose G r a d i e n t P r o f i l e o f C o n c e n t r a t e d 11S AchE which was kept i n the NMR E x p e r i m e n t a l C o n d i t i o n s 55 IV-3 S t r u c t u r e o f A c e t y l c h o l i n e s t e r a s e I n h i b i t o r s used i n NMR Experiment 57 IV-4 The XH NMR S p e c t r a o f A t r o p i n e S u l f a t e w i t h U n m o d i f i e d AchE 59 IV-5 The 1tt NMR S p e c t r a of E s e r i n e S u l f a t e w i t h C a rbamylated AchE 65 IV-6 A P l o t o f AAy f o r the C-methyl P r o t o n Resonances of E s e r i n e w i t h r e s p e c t t o E 0 / l 0 ~ K D 68 - v i i i -Page IV-7 A P l o t o f R e c i p r o c a l o f A A y f o r the C-methyl Resonances of E s e r i n e v s . V a r y i n g C o n c e n t r a t i o n s o f E s e r i n e S u l f a t e 68 IV-8 The *H NMR S p e c t r a o f T r i m e t h y l a m i n e Hydro-c h l o r i d e w i t h M o d i f i e d AchE 72 IV-9 The  lU NMR S p e c t r a o f A t r o p i n e S u l p h a t e w i t h M o d i f i e d AchE 73 IV-10 The *H NMR S p e c t r a of P h e n y l t r i m e t h y 1 C h l o r i d e w i t h M o d i f i e d AchE . 74 IV-11 A P l o t o f the R e c i p r o c a l o f A A y f o r the M e t h y l Group Resonances w i t h r e s p e c t t o V a r y i n g C o n c e n t r a t i o n s o f A t r o p i n e S u l f a t e 76 IV-12 A P l o t o f A A y f o r the M e t h y l P r o t o n Resonances of Phenyltrimethylammonium w i t h r e s p e c t t o IV-13 A P l o t of the R e c i p r o c a l of A A y f o r the M e t h y l Group Resonances of Phenyltrimethylammonium v s . C o n c e n t r a t i o n 77 T able I I - l E x p e c ted R e l a x a t i o n Times i n Presence of the C h e m i c a l Exchange when A i s Dominant S p e c i e s i n S o l u t i o n 30 IV-1 Observed L i n e B r o a d e n i n g s of NMR Resonances f o r I n h i b i t o r s I n t e r a c t i n g w i t h N a t u r a l AchE 60 - i x -ACKNOWLEDGEMENT A l t h o u g h I w o u l d l i k e t o t h a n k many o f t h e p e o p l e I e n c o u n t e r e d d u r i n g my s t a y i n t h e U . B . C . C h e m i s t r y d e p a r t m e n t , h e r e I w o u l d l i k e t o e x p r e s s s p e c i a l a p p r e c i a t i o n t o t h o s e w i t h whom I w o r k e d m o s t c l o s e l y : F i r s t , I w i s h t o e x p r e s s my s i n c e r e g r a t i t u d e t o my s u p e r v i s o r , D r . A l a n G . M a r s h a l l , f o r h i s c o n t i n u a l g u i d a n c e a n d p o s i t i v e e n c o u r a g e m e n t t h r o u g h o u t t h e c o u r s e o f t h i s w o r k . I w o u l d p a r t i c u l a r l y l i k e t o t h a n k D r . G. Webb f o r h i s g e n e r o u s h e l p and u s e f u l s u g g e s t i o n s , w i t h o u t w h i c h t h i s w o r k w o u l d n o t h a v e g o t t e n o f f t h e g r o u n d . I am a l s o h a p p y t o e x t e n d my g r a t i t u d e t o my f r i e n d s W. A p p e l , P . B u r n s , G . L u o m a , J . S m i t h a n d L . W a l s h , who p r o v i d e d e m o t i o n a l as w e l l as p h y s i c a l s u p p o r t . - 1 -CHAPTER I INTRODUCTION The work p r e s e n t e d i n t h i s t h e s i s d e s c r i b e s an i n v e s t i g a -t i o n o f the i n t e r a c t i o n between e l e c t r i c e e l a c e t y l c h o l i n e s t e r -ase (AchE) and i t s i n h i b i t o r s u s i n g NMR t e c h n i q u e s . In s p i t e o f the i n t e r e s t i n AchE r e s u l t i n g from i t s i n v o l v e m e n t i n the f u n c t i o n i n g o f nerve and muscle c e l l s , the d e t a i l e d f u n c t i o n of t h i s enzyme i s as y e t not w e l l u n d e r s t o o d compared w i t h o t h e r s e r i n e enzymes, because a s t a n d a r d way o f i s o l a t i n g and p u r i f y i n g t h i s enzyme was not e s t a b l i s h e d f o r a lon g t i m e . U n t i l r e c e n t l y , i t has a l s o not been p o s s i b l e t o o b t a i n s u f -f i c i e n t q u a n t i t i e s o f h i g h p u r i t y enzymes f o r a n a l y s i s of enzyme a t the m o l e c u l a r l e v e l . The i n t r o d u c t i o n c o n s i s t s o f a b r i e f d e s c r i p t i o n of the r o l e o f AchE i n n e u r a l t r a n s m i s s i o n , f o l l o w e d by the t e c h n i q u e f o r i s o l a t i o n and the s t r u c t u r e d e t e r m i n a t i o n o f the enzyme. The c a t a l y t i c mechanism o f the enzyme and the p r e s e n t knowledge of i t s i n t e r a c t i o n w i t h i n h i b i t o r i s then c o n s i d e r e d , f o l l o w e d by a d i s c u s s i o n o f the methods a v a i l a b l e t o i n v e s t i g a t e the dynamic a s p e c t s of the enzyme. A. ACETYLCHOLINESTERASE A c e t y l c h o l i n e s t e r a s e i s the enzyme which c a t a l y z e s the h y d r o l y s i s of a c e t y l c h o l i n e and i s w i d e l y d i s t r i b u t e d i n e x c i t a b l e membranes. - 2 -CH 0 C — O — C H 2 C H 2 — N (CH 3) 3 AchE H~0 -> CH-.COOH + HO—CH 0CH„—N(CH^) + a c e t y l c h o l i n e c h o l i n e In e a r l y s t u d i e s , the enzyme c a t a l y z i n g the h y d r p l y s e s o f c h o l i n e e s t e r s was found a s s o c i a t e d w i t h nerve and muscle t i s s u e ( 1 , 2 ) . L a t e r , enzymes h a v i n g the same f u n c t i o n were found i n many o t h e r t i s s u e s . A f t e r the d i s c o v e r y of two d i s -t i n c t t y p e s o f enzyme i n bl o o d (3) - one i n the r e d c e l l s , the o t h e r i n the serum - the k i n e t i c p r o p e r t i e s of these two enzymes became the b a s i s f o r c l a s s i f y i n g enzymes i n o t h e r t i s s u e s . A c e t y l c h o l i n e s t e r a s e , the enzyme s t u d i e d i n t h i s t h e s i s , i s the r e d c e l l type which h y d r o l y z e s a c e t y l c h o l i n e f a r more r a p i d l y than b u t y l c h o l i n e i n c o n t r a s t t o the serum t y p e . A c e t y l c h o l i n e s t e r a s e i s found i n the nervous t i s s u e s o f a l l s p e c i e s o f a n i m a l s . I t s most i m p o r t a n t f u n c t i o n i s t o c a t a l y z e the h y d r o l y s i s o f the c h o l i n e r g i c t r a n s m i t t e r , a c e t y l -c h o l i n e , o f the nerve i m p u l s e . A c e t y l c h o l i n e i s one o f the c h e m i c a l t r a n s m i t t e r s which makes i t p o s s i b l e t o t r a n s m i t nerve i m p u l s e s a c r o s s s y n a p s e s , a s p e c i f i c s t r u c t u r e between two nerve c e l l s , or a nerve c e l l and a muscle c e l l . The f o l l o w i n g i s a s c h e m a t i c diagram o f a synapse. s y n a p t i c v e s i c l e s A c e t y l c h o l i n e s t e r a s e a x o n p r e s y n a p t i membrane s y n a p t i c c l e f t p o s t s y n a p t i c membrane d i r e c t i o n o f n e r v e i m p u l s e - 3 -W i t h i n the bulbous s t r u c t u r e a t the end o f the p r e s y n a p t i c axon, t h e r e are many s m a l l v e s i c l e s each c o n t a i n i n g i n the 4 o r d e r o f 10 m o l e c u l e s o f the n e u r o t r a n s m i t t e r . A t the c h o l i n -e r g i c synapse t h i s n e u r o t r a n s m i t t e r i s a c e t y l c h o l i n e and the a r r i v a l o f a nerve impulse a t the p r e s y n a p t i c membrane causes the l i b e r a t i o n o f quanta o f a c e t y l c h o l i n e m o l e c u l e s i n t o the s y n a p t i c c l e f t . The a c e t y l c h o l i n e then d i f f u s e s a c r o s s the s y n a p t i c c l e f t t o the p o s t s y n a p t i c membrane, where i t b i n d s t o s p e c i f i c r e c e p t o r m o l e c u l e s . These r e c e p t o r m o l e c u l e s , the a c e t y l c h o l i n e r e c e p t o r s , are a s s o c i a t e d w i t h i o n c h a n n e l s . On the b i n d i n g o f a c e t y l c h o l i n e , the c h a n n e l s open and d e p o l a r i z e the p o s t s y n a p t i c membrane by p e r m i t t i n g a l a r g e inward c u r r e n t o f N a + . T h i s d e p o l a r i z a t i o n o f the p o s t s y n a p t i c membrane t r i g g e r s an a c t i o n p o t e n t i a l i n the a d j a c e n t axon or muscle membrane. The d e p o l a r i z i n g s i g n a l must be s w i t c h e d o f f q u i c k l y t o r e s t o r e the e x c i t a b i l i t y o f the p o s t s y n a p t i c membrane. T h i s i s a c h i e v e d by the e f f i c i e n t enzyme, A c e t y l c h o l i n e s t e r a s e , which i s h i g h l y c o n c e n t r a t e d a t the s u r f a c e o f the p o s t s y n a p t i c membrane. A c e t y l c h o l i n e s t e r a s e (AchE) used i n t h i s e x p e r i m e n t was pr e p a r e d from the e l e c t r i c organ o f the e l e c t r i c e e l , E l e c t r o - phorus e l e c t r i c u s . E l e c t r i c organs c o n s i s t o f columns of c e l l s c a l l e d e l e c t r o p l a x e s , which have e v o l v e d from muscle c e l l s . They r e t a i n the e x c i t a b l e o u t e r membrane o f muscle but have l o s t the c o n t r a c t i l e a p p a r a t u s . The e l e c t r i c e e l i s v e r y - 4 -r i c h i n c h o l i n e r g i c p o s t s y n a p t i c membrane, a p p r o x i m a t e l y 70% of i t s body c o n s i s t i n g o f e l e c t r i c o r g a n . For t h i s r e a s o n , the e l e c t r i c e e l i s an e x c e l l e n t source of l a r g e q u a n t i t i e s o f AchE. A l t h o u g h the most p o p u l a r source o f AchE are the e l e c -t r i c organs o f the e l e c t r i c e e l and the t o r p e d o , o t h e r s o u r c e s i n c l u d e b o v i n e e r y t h r o c y t e s , f l y head and human b r a i n . ( I t i s thus e v i d e n t t h a t AchE may occur i n t i s s u e s w i t h o u t b e i n g a s s o c i a t e d w i t h nerves.) However, AchE from d i f f e r e n t s o u r c e s may show a v a r i a t i o n i n r e a c t i v i t y toward i r r e v e r s i b l e i n h i -b i t o r s ( 4 ) . For example, p h e n y l e t h y l s u l f o n y l f l u o r i d e i n h i b i t s b o v i n e e r y t h r o c y t e AchE, but does not i n h i b i t (at a measurable r a t e ) AchE from the e l e c t r i c organ ( 5 , 6 ) . In s p i t e o f th e s e r e p o r t e d d i f f e r e n c e s , the f i n d i n g s f o r AchE from one source s h o u l d be a p p l i c a b l e t o the enzymes from o t h e r s o u r c e s , due t o the overwhelming s i m i l a r i t i e s i n the enzyme s t r u c t u r e , the sub-s t r a t e and i n h i b i t o r s p e c i f i c i t y , and the t u r n o v e r numbers o f a c e t y l c h o l i n e ( 7 ) . B. EXTRACTION AND PURIFICATION The appearance o f a heterogeneous s i z e d i s t r i b u t i o n o f AchE i n h i g h i o n i c s t r e n g t h e x t r a c t s o f f r e s h e e l t i s s u e was f i r s t r e c o g n i z e d by M a s s o u l i e and R i e g e r ( 8 ) . They c h a r a c t e r -i z e d the m o l e c u l a r forms o f AchE (by t h e i r s e d i m e n t a t i o n c o e f f i c i e n t ) t o be the 9S, 14S and 18S s p e c i e s . Other workers o b t a i n e d a m i x t u r e o f AchE c o n s i s t i n g m o s t l y o f the l i s form - 5 -(9,10). I t i s now s p e c u l a t e d t h a t the 18S form o f AchE i s c r e a t e d a t the synapses from s m a l l e r b u i l d i n g b l o c k s o f the 9S form o f AchE, and a l l t h r e e forms, 9S, 14S and 18S c o e x i s t a t the s u r f a c e o f the p o s t s y n a p t i c membrane (11). I t i s a l s o known t h a t p r o t e o l y s i s d u r i n g the s o l u b i l i z a t i o n p r o c e s s can c r e a t e s m a l l e r forms o f 9S, 11S and 14S from the 18S form of AchE (11,12) . However, the importance o f the e x t r a c t i o n procedure t o the subsequent c h a r a c t e r i z a t i o n o f the i s o l a t e d enzyme was not r e c o g n i z e d u n t i l about 1970 (13), and t h i s l e d t o the d i v e r -s i t y o f the AchE s p e c i e s t h a t were i s o l a t e d from e l e c t r i c organs (10,14-16). H i s t o r i c a l l y two ty p e s of e x t r a c t i o n p r o -cedure have been used f o r the s o l u b i l i z a t i o n o f the enzyme from e e l e l e c t r i c o r g a n , which produced w e l l d e f i n e d AchE d i s t r i b u -t i o n s . The f i r s t p r o c e d u r e i n v o l v e s e x p o s i n g the enzyme t o a p r o t e o l y t i c agent, or immersing the e e l t i s s u e i n t o l u e n e ; t h i s y i e l d s the l i s form o f AchE (7). In the second p r o c e d u r e , the e l e c t r i c organ i s homogenized a t h i g h i o n i c s t r e n g t h , which produces a m i x t u r e o f 9S, 14S and 18S forms of AchE as mentioned p r e v i o u s l y . A f f i n i t y chromatography developed r e c e n t l y has reduced the p u r i f i c a t i o n o f AchE t o a much s i m p l e r o n e - s t e p o p e r a t i o n (11,17). T h i s t a k e s advantage o f the range o f s m a l l o r g a n i c i n h i b i t o r s which b i n d t o s p e c i f i c s i t e s o f AchE. One of t h e s e i n h i b i t o r l i g a n d s i s c o v a l e n t l y c o u p l e d and i m m o b i l i z e d on a r e s i n m a t r i x . When a crude enzyme s o l u t i o n permeates - 6 -through r e s i n m a t r i x , the i m m o b i l i z e d l i g a n d s i n t e r a c t w i t h the enzyme and causes a p r e f e r e n t i a l r e t a r d a t i o n , the b i n d i n g a f f i n i t y f o r l i g a n d s t o the enzyme v a r y i n g g r e a t l y w i t h the i o n i c s t r e n g t h o f the medium. The enzyme used i n t h i s t h e s i s was e x t r a c t e d i n a h i g h i o n i c s t r e n g t h medium, a c c o r d i n g t o the second p r o c e d u r e . Subsequent procedure i n v o l v e d an a f f i n i t y m a t r i x w i t h the N - m e t h y l a c r i d i n i u m i o n (see F i g u r e 1-5) as a l i g a n d , w hich i s c a p a b l e o f b i n d i n g t o the enzyme even a t a h i g h i o n i c s t r e n g t h (17,19). C. STRUCTURE A c e t y l c h o l i n e s t e r a s e i s o l a t e d from the e l e c t r i c organ t i s s u e o f the e l e c t r i c e e l can be o b t a i n e d i n a number o f d i f -f e r e n t m o l e c u l a r forms. Three forms can be e x t r a c t e d a t h i g h i o n i c s t r e n g t h from f r e s h t i s s u e , w i t h s e d i m e n t a t i o n c o e f f i -c i e n t s o f 18S, 14S and 9S, and a l l appear h i g h l y asymmetric (20 ) . These s p e c i e s aggregate a t low i o n i c s t r e n g t h (11, 18, 2 1 ) , and d e t a i l e d s t u d i e s o f the s e s p e c i e s were thus p o s s i b l e o n l y a f t e r the i n t r o d u c t i o n o f a f f i n i t y chromatography w i t h the a p p r o p r i a t e l i g a n d s f o r p u r i f i c a t i o n a t h i g h i o n i c s t r e n g t h . The 11S s p e c i e s , however, a d e g r a d a t i o n p r o d u c t from p r o t e o l y s i s o f the n a t i v e enzyme (8,11), does not aggregate even a t v e r y low i o n i c s t r e n g t h s (18,22). P u r i f i c a t i o n of t h i s s p e c i e s t h e r e -f o r e , was a c h i e v e d much e a r l i e r than o f the 9S, 14S and 18S - 7 -forms, and c o n s e q u e n t l y s t u d i e d i n g r e a t e r d e t a i l . The l i s form o f the enzyme can be produced e i t h e r by endogenous p r o t e -o l y s i s d u r i n g s t o r a g e or by t r e a t i n g e x t r a c t s w i t h v a r i o u s p r o t e a s e s . S i l m a n d e s c r i b e d a s c h e m a t i c model of 18S e e l AchE (23) based on d e t a i l e d s t u d i e s o f the p r o t e o l y t i c c l e a v a g e and o t h e r p h y s i c o c h e m i c a l d a t a t o g e t h e r w i t h e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s on the asymmetric forms of AchE (25,26). T h i s model i s v e r y s i m i l a r t o the one proposed e a r l i e r by R o s e n b e r r y (24) , e x c e p t t h a t the t a i l i s e x p r e s s e d as a t r i p l e h e l i x , and i s shown i n F i g u r e I - l . S i m p l i f i e d models of m u l t i p l e forms of AchE i n F i g u r e 1-2 i l l u s t r a t e the d i f f e r e n t s p e c i e s o f AchE (2 7 ) , which are t y p i c a l l y i s o l a t e d by h o m o g e n i zation of e e l e l e c t r i c t i s s u e i n the presence o f 1-2 M NaCl and the c h e l a t -in g a gent. i ) The 9S, 14S and 18S S p e c i e s Three forms of n a t i v e AchE w i t h s e d i m e n t a t i o n c o e f f i c i e n t s o f a p p r o x i m a t e l y 9S, 14S and 18S, behave h y d r o d y n a m i c a l l y as asymmetric p a r t i c l e s (20,28). The unique e l o n g a t e d s t r u c t u r e of a l l t h r e e forms, i n which a m u l t i - s u b u n i t head i s connected t o a t a i l , i s seen under the e l e c t r o n microscope (25,26), and the s p e c i e s p i c t u r e i n F i g u r e 1-2 are i d e n t i f i e d . W h i l e the t a i l i s o f a p p r o x i m a t e l y the same l e n g t h , the heads o f 9S, 14S and 18S forms c o n t a i n one, two and t h r e e t e t r a m e r s o f c a t a l y t i c s u b u n i t s r e s p e c t i v e l y ( 2 9 ) . The i n d i v i d u a l t e t r a m e r , which i s the g l o b u l a r form i s o l a t e d f o l l o w i n g p r o t e o l y t i c d i g e s t i o n , - 8 -lQ;!t(J V^-Q Os-sO/ t r y p s i n 1 J c o l l a g e n a s e - S - S -- S - S -> F i g u r e 1 - 1 : M o d e l o f t h e 18S a c e t y l c h o l i n e s t e r a s e . P o i n t s o f c l e a v a g e by s o d i u m d o d e c y l s u l f a t e , t r y p s i n a n d c o l l a g e n a s e a r e shown by a r r o w s . - 9 -f o r m s e d i m e n t a t i o n c o e f f i c i e n t m o l e c u l a r w e i g h t ( t o t a l ) ( t a i l ) OO OO OO \ OO o o v / o o 18.4S i , i 50 5ooo 157,000 o o OO O O V D O 14.2 S 796,000 134,000 OO QO 9.2 S 410,000 79,000 $3 11.8S 3 3 1 >000 F i g u r e 1 - 2 : M u l t i p l e f o r m s o f A c e t y l c h o l i n e s t e r a s e f r o m E . E l e c t r i c u s . The b e s t d e t e r m i n e d v a l u e s f o r b o t h s e d u m e n t a t i o n c o e f f i -c i e n t and m o l e c u l a r w e i g h t a r e s h o w n . M o l e c u l a r w e i g h t o f t a i l s a r e shown as t h e d i f f e r e n c e b e t w e e n t h e t o t a l MW a n d t h e sum o f t h e MM o f t h e t e t r a m e r i c u n i t p r e s e n t . - 10 -the symmetric form, i s known as the l i s s p e c i e s . The c a t a l y t i c p r o p e r t i e s o f d i f f e r e n t s p e c i e s o f AchE, w i t h the e x c e p t i o n o f the aggregated forms appear t o be e s s e n t i a l l y i d e n t i c a l (29,30), as i s t h e i r b e h a v i o r towards i n h i b i t o r s (31). The p r i n c i p a l form, 18S AchE, w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 1,150,000 c o n t a i n s 12 c a t a l y t i c s u b u n i t s i n i t s head arranged o i n t h r e e t e t r a m e r i c g r o u p s , and has a t a i l a p p r o x i m a t e l y 500 A l o n g . Each of the t h r e e s u b u n i t t e t r a m e t e r s , as seen from F i g u r e I - l , i s l i n k e d by a d i s u l f i d e bond t o one s t r a n d o f a t r i p l e h e l i x . In each t e t r a m e r , the s u b u n i t dimers t h a t are not c o v a l e n t l y l i n k e d t o the t a i l may be detached by sodium d o d e n y l s u l f a t e (23,24). T r y p s i n c l e a v e s the 18S form of AchE t o d i s c r e t e t e t r a m e r s by a t t a c k i n g the neck of the t r i p l e h e l i x . On the o t h e r hand, c o l l a g e n a s e produces a 20S form, c o n s i s t i n g o f heads w i t h a r e s i d u a l t a i l h o l d i n g the t e t r a m e r s t o g e t h e r , by a t t a c k i n g the m i d p o i n t o f the t r i p l e h e l i x (23,32,33). E i t h e r o f these c o n v e r s i o n s d e s t r o y s the c h a r a c t e r i s t i c c a p a c i t y f o r s e l f - a s s o c i a t i o n , which i s seen i n the 18S form as w e l l as the 14S and 9S forms a t low i o n i c s t r e n g t h ( 3 2 ) . S i n c e o n l y the t a i l e d forms of e l e c t r i c organ AchE are a g g r e g a t e d , the t a i l i s c o n s i d e r e d t o be r e s p o n s i b l e f o r the a g g r e g a t i n g p r o p e r t y (32, 3 4 ) . T h i s i s suggested by e l e c t r o n m i c r o s c o p y o f the m u l t i -m o l e c u l a r a s s e m b l i e s i n which the t a i l s are packed s i d e - t o - s i d e i n b u n d l e s (34,35). T h i s r o d - l i k e t a i l i s now c o n s i d e r e d a k i n t o c o l l a g e n . T h i s s u p p o s i t i o n i s s u p p o r t e d by: 1., the s i m i l a r i t y - 11 -i n amino a c i d c o m p o s i t i o n ( 2 4 ) , 2., the t h r e e - s t r a n d e d appear-ance as seen i n e l e c t r o n m i c r o s c o p y ( 2 6 ) , and 3., the c h a r a c -t e r i s t i c m o d i f i c a t i o n by c o l l a g e n a s e . There i s an observed c l o s e s i m i l a r i t y i n c o m p o s i t i o n o f the t a i l and basement mem-brane s . T h i s s u g g e s t s t h a t the c o l l a g e n - l i k e t a i l i s d e r i v e d from basement membrane c o l l a g e n w i t h i n the s y n a p t i c gap, and f u n c t i o n s i n the i m m o b i l i z a t i o n of the enzyme (23,27,33,36). i i ) The 11S S p e c i e s As d i s c u s s e d p r e v i o u s l y , a u t o l y s i s and t r e a t m e n t w i t h v a r i o u s p r o t e a s e s o f e l e c t r i c organ t i s s u e e x t r a c t s l e d t o the appearance o f an a d d i t i o n a l m o l e c u l a r form, a g l o b u l a r 11S AchE ( F i g u r e 1-2) . T h i s d e g r a d a t i o n appears t o i n v o l v e the s e q u e n t i a l removal of one 11S t e t r a m e r from the 18S form t o g i v e the 14S s p e c i e s , and o f a second 11S t e t r a m e r t o g i v e the 9S s p e c i e s . The 11S s p e c i e s so formed l a c k s a t a i l and con-t a i n s f o u r monomers, the s m a l l e s t c a t a l y t i c s u b u n i t s , w i t h m o l e c u l a r w e i g ht 80,000 ( 3 7 ) , which appear t o be i d e n t i c a l s t r u c t u r a l l y but assembled w i t h s l i g h t asymmetry ( 3 8 ) . When a l i g a n d i n t e r a c t s w i t h the enzyme, the s t o i c h i o m e t r y i s 1:1 w i t h a 80,000 d a l t o n s u b u n i t on the t e t r a m e r i c 11S enzyme ( 3 9 ) . The c a t a l y t i c p r o p e r t i e s and b e h a v i o r toward i n h i b i t o r s o f the c a t a l y t i c a l l y a c t i v e monomers show no d i f f e r e n c e from the 18S, 14S and 11S enzyme (37,38). The 80,000 d a l t o n s u b u n i t , t h e r e -f o r e , can be c o n s i d e r e d as an independent u n i t o f the enzyme w i t h r e s p e c t t o the i n t e r a c t i o n w i t h l i g a n d s . T h i s monomer i s - 12 -s u s c e p t i b l e t o p r o t e o l y t i c c l e a v a g e a t a s p e c i f i c s i t e and a l l p r o t e a s e s e x c e p t c o l l a g e n a s e (40) produce the 11S form which g e n e r a t e s a 50,000 d a l t o n fragment c o n t a i n i n g an a c t i v e s i t e and minor fragments c o n t a i n i n g no a c t i v e s i t e s (41) . T h i s p r o -t e o l y s i s o f c a t a l y t i c s u b u n i t s does not r e l e a s e fragments from the 11S enzyme but i s d e t e c t a b l e o n l y a f t e r the enzyme i s d e n a t u r e d and s u b j e c t e d t o d i s u l f i d e bond r e d u c t i o n (12,40). A l t h o u g h the c o n v e r s i o n o f the 18S and 14S m o l e c u l a r forms t o a g l o b u l a r 11S form o c c u r s f a s t e r than p r o t e o l y t i c c l e a v a g e , the e x t e n t o f the c l e a v a g e v a r i e s depending on the c o n d i t i o n s of p u r i f i c a t i o n and s t o r a g e . However, t h i s p r o t e o l y s i s which fragments a p o r t i o n of the c a t a l y t i c s u b u n i t has no apparent e f f e c t on the c a t a l y t i c p r o p e r t i e s of the enzyme (38 ) . D. CATALYTIC MECHANISM A c e t y l c h o l i n e s t e r a s e i s c l a s s i f i e d as a s e r i n e h y d r o l a s e t o g e t h e r w i t h o t h e r e s t e r a s e s and p e p t i d a s e s , which are r e a d i l y i n h i b i t e d i r r e v e r s i b l y by p h o s p h o r y l a t i o n a t the s e r i n e r e s i d u e a c t i v e s i t e . The amino a c i d sequence about t h i s r e s i d u e shows s i g n i f i c a n t s i m i l a r i t i e s t o the enzymes o f t h i s c l a s s (42). S e v e r a l s e r i n e h y d r o l a s e s i n t h i s c l a s s show a s i m i l a r i t y i n t h e i r 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 s as d e t e r m i n e d by X-ray c r y s t a l l o g r a p h y (43) . In the absence of X-ray d i f f r a c t i o n d e t e r m i n a t i o n o f the 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 AchE, i n -f e r e n c e s about mechanism so f a r have been based on both - 13 -s u b s t r a t e c a t a l y s i s and a n a l o g i e s drawn from c h y m o t r y p s i n w i t h r e s e r v a t i o n s , due t o i m p o r t a n t d i f f e r e n c e s i n the k i n e t i c s . i ) S u b s t r a t e R e a c t i o n The a c t i v e s i t e of the enzyme, a c e t y l c h o l i n e s t e r a s e , con-s i s t s o f two p r i n c i p a l s u b s i t e s - an a n i o n i c s i t e and an e s t e r -a t i c s i t e (44) . The a n i o n i c s i t e i s r e s p o n s i b l e f o r b i n d i n g and o r i e n t i n g ammonium i o n , wheras the e s t e r a t i c s i t e i s i n v o l v e d i n the a c t u a l c a t a l y t i c p r o c e s s . Recent s t u d i e s show t h a t the p r e s ence o f i n h i b i t o r s o r c h e m i c a l m o d i f i c a t i o n o f enzyme a t the a c t i v e s i t e a f f e c t s the r a t e of h y d r o l y s i s of v a r i o u s s u b s t r a t e s d i f f e r e n t l y . T h i s i m p l i e s t h a t t h e r e i s more than one b i n d i n g s i t e i n the a c t i v e r e g i o n . However f o r the p r e s e n t p u r p o s e , the s i m p l e two p o c k e t model shown i n F i g u r e 1-3 i s a p p r o p r i a t e . The m e c h a n i s t i c scheme i s g i v e n i n the same f i g u r e (45-47). I n i t i a l l y , a c e t y l c h o l i n e (Ach) r e a c t s w i t h the enzyme a t i t s a c t i v e s u r f a c e f o r m i n g a M i c h a e l i s -Menten complex. Most o f the b i n d i n g of Ach i s accounted f o r by Coulombic i n t e r a c t i o n s a t the a n i o n i c s i t e (48). W ith the a i d o f i m i d a z o l e , Ach undergoes n u c l e o p h i l i c a t t a c k by the s e r i n e r e s i d u e o f AchE, d i s p l a c i n g c h o l i n e but l e a v i n g the a c e t a t e m o i e t y c o v a l e n t l y bound t o the e s t e r a t i c s i t e . The a c y l enzyme so formed i s h y d r o l y z e d e f f i c i e n t l y t o r e g e n -e r a t e the a c t i v e s u r f a c e . T h i s d e a c e t y l a t i o n i s the r a t e l i m i t i n g s t e p f o r a good s u b s t r a t e , such as Ach ( 4 9 ) . A t h i g h s u b s t r a t e c o n c e n t r a t i o n s , the h y d r o l y s i s i s Acetylcholine - 1 4 -hydrolysis E + S ^ E - S — - > EA+ P—> E + P I 2 . a n i o n i c s i t e e s t e r a t i c s i t e s e r y l F i g u r e 1 - 3 : The r e a c t i o n squeme o f t h e h y d r o l y s i s o f a c e t y l c h o l i n e by a c e t y l c h o l i n e s t e r a s e . E , e n z y m e ; S , s u b s t r a t e ; E S , M i c h a e l i s - M e n t e n c o m p l e x ; Pt . c h o l i n e ; P 2 » a c e t i c a c i d ; E A , a c e t y l e n z y m e . - 15 -inhibited by the binding of a second substrate molecule to the acyl enzyme EA (50) . The mechanism of this i n h i b i t i o n forming a complex EAS has wide applications. The action of reversible i n h i b i t o r s and substrate reactions i s generally explained in terms of binding to the acylated as well as to the free enzyme. i i ) Inhibitors and Their Interaction with the Enzyme Acetylcholinesterase i s now considered to possess, in addition to the anionic s i t e in the active center, a peripheral anionic s i t e where ligands bind and exert a regulatory influence on the enzyme a c t i v i t y . S i g n i f i c a n t evidence for the existence of t h i s s i t e has been obtained from ki n e t i c studies using natural and synthetic substrates with various i n h i b i t o r s (21, 51,52), and more recently through nuclear magnetic resonance (53) and fluorescence spectroscopic measurements of ligand association with the enzyme (39,54). Bisquaternary ligands having two ca t i o n i c groups are considered to span two anionic s i t e s and the distance between two s i t e s can be estimated at o 14 A, a distance of 10 carbon atoms apart (52,54,55). The decamethonium ion, with two quaternary ammonium groups sepa-rated by a 10 carbon a l k y l chain has thus a high a f f i n i t y to-wards AchE, and i s used for eluting AchE during p u r i f i c a t i o n , by replacing the N-methylacridinium ion i n i t i a l l y bound to the enzyme. The binding modes of these ligands to AchE i s shown schematically in Figure 1-4 (27,55). - 16 -a c t i v e c e n t e r e s t e r a t i c s i t e a n i o n i c s i t e O ft C H . — C — 0 — C H 9 — C H 9 — N ( C H , ) , 3 II c c. | 5 i a c e t y l c h o l i n e + N ( C H J 3 ' 3 N - m e t h v l a c r i d i n i u m 1 p e r i p h e r a l a n i o n i c s i t e ( C H 2 ) , 0 + N ( C H 3 ) 3 d e c a m e t h o n i u m F i g u r e 1 - 4 : B i n d i n g s i t e s o f a c e t y l c h o l i n e s t e r a s e . W e l l r e c o g n i z e d b i n d -i n g s i t e s o f A c h E a r e shown w i t h a c e t y l c h o l i n e as a s u b s t r a t e , as w e l l as l i g a n d s u s e d f o r A c h E p u r i f i c a t i o n . The a n g u l a r o r i e n t a t i o n among t h e s i t e s a n d t h e h y d r o p h o b i c r e g i o n , how-e v e r , i s n o t k n o w n . D e c a m e t h o n i u m i s shown t o b i n d t o b o t h a n i o n i c s i t e s , d i s p l a c i n g t h e l i g a n d s o c c u p y i n g e i t h e r s i t e ( 3 9 ) . The d i s t a n c e b e t w e e n two a n i o n i c s i t e s i s d e d u c e d f r o m o p t i m a l b i n d i n g i n t h e s e r i e s o f b i s q u a t e r n a r y l i g a n d s s u c h as p o l y m e t h y l e n e - b i s - t r i m e t h y l a m m o n i u m a n d p o l y e t h y l e n e - b i s -q u i n o l i n i u m . x C r a m c l a i m s t h a t t h e N - m e t h y l a c r i d i n i u m i o n a l s o b i n d s t h e p e r i p h e r a l s i t e ( 4 2 ) . - 17 -AchE i n h i b i t o r s are t r a d i t i o n a l l y divided into two groups depending on their mode of action. The f i r s t group consists of reversible i n h i b i t o r s , and which react with AchE to form an equilibrium complex according to the following scheme. E + I g4V>EI Almost a l l reversible i n h i b i t o r s contain at least one p o s i t i v e l y charged nitrogen group, which binds to the negatively charged anionic s i t e on AchE i n h i b i t i n g i t s a c t i v i t y . They vary in size from simple i n h i b i t o r s such as divalent cations and t e t r a -alkylammonium compounds, to large nueromuscular blocking drugs such as d-tubocurarine and gallamine (see Figure 1-5). These i n h i b i t o r s should be useful tools in investigating the func-t i o n a l properties of the peripheral s i t e once their binding s i t e s (either of the two anionic sites) are p o s i t i v e l y i d e n t i -f i e d . This has proved to be a d i f f i c u l t task, and only some of the i n h i b i t o r binding s i t e s are i d e n t i f i e d with some certainty. The triethylammonium and propidium ions, and divalent inorganic cations bind to the peripheral anionic s i t e (54-57), whereas the 3-hydroxyphenyltrimethyl ammonium (HPTA) and edrophonium ions (see Figure 1-5) bind to the active anionic s i t e (51,54 ,58). How-ever, reports on i n h i b i t o r s such as N-methylacridinium, Tetra-methyl ammonium (TeMA), gallamine and d-tubocurarine d i f f e r as to which anionic s i t e s are involved (perhaps both) (51,54,56, 59-61). Even among the ligands which seem to occupy the same s i t e s , a simple competitive relationship for occupation of a - 18 -N - m e t h y l a c r t d i n t u m 0-(CH 2)—N-(C 2H 5) 3 0—(CH 2)—N—(C 2H 5) 3 N C H 2 ) 2 — N — ( C 2 H 5 ) 3 g a l 1 a m i n e (CH 3) 2 d - t u b o c u r a r i n e F i g u r e 1 - 5 : S t r u c t u r e o f A c h E i n h i b i t o r s - 19 -single s i t e does not always exist (54). Roufogalis (55) and Abou-Donia (56) proposed a second peripheral binding s i t e for gallamine. This confusion resulted from most studies being r e s t r i c t e d to inferences based on steady-state kinetics of the hydrolysis of substrates in the presence of i n h i b i t o r s . The kinetics behavior of AchE and the binding of i n h i b i t o r s are also affected by the ionic strength (39 ,54 ,62-64) , pH (51), the source of AchE (65) and aging (46). The s e n s i t i v i t y of AchE to ionic strength led Mooser to proposed the existence of two conformationally d i s t i n c t forms of s o l u b i l i z e d enzyme (39), which was later supported by Taylor (66). The second group i s comprised of i r r e v e r s i b l e i n h i b i t o r s which are esters or acid halides of phosphoric, carbamic and sulfonic acids. They react by a scheme analogous to substrate hydrolysis. The acid moiety of the i n h i b i t o r i s transferred to the e s t e r a t i c subsite forming an acyl-enzyme intermediate which, unlike the acetyl-enzyme formed with substrates, i s r e l a t i v e l y stable to hydrolysis. The h a l f - l i v e s for the hydrolyses of the carbamyl-enzymes are of the order of minutes, for phosphoryl-enzymes of the order of hours, and for the methanesulfonyl-enzymes at rates that are too slow to be measured (67) . The strong i n h i b i t o r y power of these acid-transferring i n h i b i t o r s has found a popular application in a g r i c u l t u r a l i n s e c t i c i d e s , and as nerve gases,. . Parathion and 1-naphthyl-N-methyl car-bamate (carbaryl), are used as i n s e c t i c i d e s . Tabum and Sarin, - 20 -c a u s i n g r e s p i r a t o r y p a r a l y s i s , were used as war gases d u r i n g the Second World War. Hence the development of an ant i d o t e , a n u c l e o p h i l i c r e a c t i v a t o r , has l o n g been of r e s e a r c h i n t e r e s t . R e v e r s i b l e i n h i b i t o r s have been r e c e n t l y found t o a c c e l e r a t e the spontaneous r e a c t i v a t i o n o f c arbamyl and p h o s p h o r y l AchE (47, 50,51,62,64). S u l f o n y l a t i o n by m e t h a n e s u l f o n y l f l u o r i d e i s a c c e l -e r a t e d by some c a t i o n i c i n h i b i t o r s (TeMA, TeEA, and N-methyl-p i r i d i n i u m ) , but d e c e l e r a t e d by o t h e r s (HPTA and g a l l a m i n e ) (47,68). These o b s e r v a t i o n s t o g e t h e r w i t h the s y n e r g i s t i c e f f e c t s seen on a c e t y l c h o l i n e h y d r o l y s i s by p a i r s o f i n h i b i -t o r s , are now c o n s i d e r e d by s e v e r a l a u t h o r s t o r e f l e c t c o n f o r -m a t i o n a l changes induced by the b i n d i n g of c a t i o n i c i n h i b i t o r s (21,47,51,58 ,69,70) . E. SPECTROSCOPIC TECHNIQUES IN THE STUDY OF THE CATIONIC  MECHANISM OF ACETYLCHOLINESTERASE Most i n f o r m a t i o n on the b i n d i n g of c a t i o n i c i n h i b i t o r s and hence on the c a t a l y t i c mechanism, has been d e r i v e d from e i t h e r t h e i r i n h i b i t o r y e f f e c t on the enzyme a c t i v i t y , or t h e i r i n -f l u e n c e on a c y l a t i o n or d e a c y l a t i o n o f the a c t i v e s i t e s e r i n e by an a c i d - t r a n s f e r r i n g group. However, t h e r e are a m b i g u i t i e s i n t h i s k i n e t i c approach, as the i n t e r p r e t a t i o n o f r e s u l t s i s h i n d e r e d by the p r e f e r e n c e o f two b i n d i n g s i t e s f o r c a t i o n i c l i g a n d s and a c h a r a c t e r i s t i c two s t e p c a t a l y t i c mechanism. - 21 -S p e c t r o s c o p i c t e c h n i q u e s p r o v i d e a u s e f u l t o o l f o r s t u d y i n g the i n t e r a c t i o n between i n h i b i t o r s and AchE, these b e i n g : 1) n u c l e a r magnetic res o n a n c e , 2) f l u o r e s c e n c e s p e c t r o s c o p y , and 3) s p i n l a b e l l i n g by e l e c t r o n s p i n resonance. The 1E n u c l e a r magnetic resonance (NMR) t e c h n i q u e t o date has been used f o r AchE o n l y by Kato (71-74). T h i s method g i v e s d e t a i l e d i n f o r m a t i o n on most i n h i b i t o r s i n t e r a c t i n g w i t h the enzyme. Two t y p e s o f changes are commonly o b s e r v e d . F i r s t , the l i n e w i d t h s o f i n h i b i t o r p r o t o n s r e f l e c t the degree of r e s t r i c t i o n o f motion of t h e i r s p e c i f i c p a r t s . Second, changes i n c h e m i c a l s h i f t p r o v i d e i n f o r m a t i o n on the magnetic environment when bound. The degree o f s a t u r a t i o n o f b i n d i n g s i t e s and r a t e s o f exchange may a l s o be o b t a i n e d . The resonance s i g n a l o f bound i n h i b i t o r s can not however be seen d i r e c t l y because of t h e i r s m a l l p o p u l a t i o n compared w i t h f r e e i n h i b i t o r s . Hence, the i n f o r m a t i o n p r e v i o u s l y mentioned may be o n l y de-duced under c e r t a i n c o n d i t i o n s o f the system. T h i s r e l a t i o n -s h i p w i l l be d i s c u s s e d i n d e t a i l i n Chapter I I . A t r o p i n e and e s e r i n e were used by Kato t o s t u d y the e f f e c t of pH and i o n i c s t r e n g t h , as w e l l as the presence of i r r e v e r s i b l e and r e v e r s i b l e i n h i b i t o r s on b i n d i n g . He r e p o r t e d however t h r e e d i f f e r e n t l i n e w i d t h s f o r bound i n h i b i t o r s . For example, the l i n e w i d t h of a t r o p i n e C-methyl p r o t o n i s r e p o r t e d t o be 21,000 Hz ( 7 1 ) , 5,600 Hz (72) and 984 Hz ( 7 4 ) . I t w i l l a l s o be shown i n Chapter IV t h a t the maximum p o s s i b l e l i n e w d i t h o f bound - 22 -i n h i b i t o r f o r 11S AchE i s 400 Hz. The reason f o r Kato's c o n f l i c t -i n g r e s u l t s w i l l be d i s c u s s e d i n Chapter V, t o g e t h e r w i t h the r e s u l t s o b t a i n e d i n the p r e s e n t work. F l u o r e s c e n c e s p e c t r o s c o p y has been g a i n i n g p o p u l a r i t y i n s t u d y i n g l i g a n d a s s o c i a t i o n w i t h AchE. The m o n i t o r i n g o f l i g a n d - A c h E complex f o r m a t i o n by f l u o r e s c e n c e r e q u i r e s l i g a n d s which e i t h e r e x h i b i t g r e a t l y d i m i n i s h e d quantum y i e l d s upon a s s o c i a t i o n w i t h the enzyme (39,54,75), or whose a b s o r p t i o n s p e c t r a are s u i t a b l e t o quench p r o t e i n t r y p t o p h a n y l f l u o r e s -cence upon b i n d i n g t o the enzyme (66,76). The b i n d i n g s i t e s o f s e v e r a l f l u o r e s c e n t probes have been w e l l e s t a b l i s h e d . E d r o -phonium and N - m e t h y l a c r i d i n i u m show a marked p r e f e r e n c e f o r b i n d i n g t o t h e a c t i v e c e n t e r ( 3 9 ) , whereas p r o p i d i u m a t low i o n i c s t r e n g t h b i n d s e x c l u s i v e l y t o the p e r i p h e r a l a n i o n i c s i t e ( 5 4 ) . These l i g a n d s are now used i n f l u o r e s c e n c e t i t r a -t i o n t o s t u d y the b i n d i n g p r o p e r t i e s and i n t e r a c t i o n o f o t h e r n o n - f l u o r e s c e n t q u a t e r n a r y ammonium l i g a n d s . The f l u o r e s c e n c e t e c h n i q u e , as o t h e r s p e c t r o s c o p i c methods, p r o v i d e s a more c o n v e n i e n t measurement o f k i n e t i c p a r a m e t e r s , s i n c e the l i g a n d o f i n t e r e s t can be d i r e c t l y o b s e r v e d . For example, t h i s method e n a b l e s a d i r e c t m o n i t o r i n g o f the i n f l u e n c e o f the l i g a n d on the m o d i f i c a t i o n o f the enzyme by a c i d - t r a n s f e r r i n g g r o u p s . T h i s was t r a d i t i o n a l l y o b t a i n e d by measuring r e s i d u a l enzyme a c t i v i t y f o l l o w i n g e x t e n s i v e d i l u t i o n of the r e a c t a n t m o l e c u l e (7 6 ) . In the s t u d y o f the a c y l a t i o n o f AchE, t h e r e are two - 23 -k i n e t i c parameters which are o f t e n d i f f i c u l t t o s e p a r a t e . These are 1) d i s s o c i a t i o n c o n s t a n t f o r the ester-enzyme com-p l e x , and 2) the s p e c i f i c r a t e c o n s t a n t f o r the f o r m a t i o n o f acyl-enzyme f o r the complex. T h i s can be a c h i e v e d more s i m p l y by u s i n g a s u b s t r a t e w i t h a f l u o r e s c e n t l e a v i n g group ( 7 5 ) . The a s s o c i a t i o n and d i s s o c i a t i o n r a t e o f a l i g a n d b i n d i n g t o AchE may a l s o be o b t a i n e d e i t h e r by . s t o p p e d - f l o w measurements o f f l u o r e s c e n c e ( 6 6 ) , or by temperature-jump r e l a x a t i o n k i n e t i c s of f l u o r e s c e n t l i g a n d s (77) . A l t h o u g h t h e r e are a number of s p i n - l a b e l l i n g r e a g e n t s a v a i l a b l e f o r s u l f o n y l a t i n g , a c y l a t i n g and p h o s p h o r y l a t i n g (78)/ none o f them ( w i t h one e x c e p t i o n ) has been used w i t h AchE t o d a t e . The a p p l i c a t i o n o f s p i n - p r o b i n g t o AchE has been l i m i t e d t o s p i n - l a b e l l e d a n a l o g s o f the r e v e r s i b l e i n h i b i t o r s and a c e t y l c h o l i n e , a l l o f which c o n t a i n a n i t r o x i d e group, t h i s f r e e r a d i c a l b e i n g s t a b l e i n aqueous s o l u t i o n . S p i n - l a b e l l e d a c e t y l c h o l i n e a n a l o g s o f d i f f e r e n t s i z e were p r e p a r e d by Abou-Donia e t a l (55,56), and t h e i r k i n e t i c parameters o f h y d r o l y s i s s t u d i e d . ESR s t u d i e s on th e s e i n d i c a t e d t h a t the a c t i v e s u r -f a c e o f AchE has a r e l a t i v e l y open b i n d i n g s i t e , and has been r e p o r t e d e l s e w h e r e ( 7 8 ) . Wee and S i n h a observed t h a t both n i t r o x i d e groups o f s p i n - l a b e l l e d b i s q u a t e r n a r y ammonium l i g a n d s become i m m o b i l i z e d and s p i n - s p i n i n t e r a c t i o n was a b o l i s h e d upon b i n d i n g t o AchE. T h i s p r o v i d e d e v i d e n c e of b i n d i n g i n an ex-tended c o n f o r m a t i o n v i a a 2 p o i n t attachment i n v o l v i n g both - 24 -quaternary nitrogens (79). The method of s p i n - l a b e l l i n g to study substrate and/or in h i b i t o r interactions with the enzyme, provides the same type of information as NMR, the spectrum being affected by r o t a t i o n a l motional constraints and the mag-netic and e l e c t r i c environments. One advantage of ESR how-ever, as compared with NMR, i s a greater s e n s i t i v i t y . It i s thus possible to calculate the distance between the two s i t e s on the enzyme u t i l i z i n g one of the following: 1) two spin labels, 2) a spin l a b e l with a diamagnetic nucleus, or 3) a spin l a b e l with a paramagnetic ion. The high s e n s i t i v i t y of ESR also means that r e l a t i v e l y small concentrations of i n h i b i -tor can be used, resulting in a higher f r a c t i o n of bound i n h i -b i t o r , which enables a d i r e c t observation of the bound i n h i b i t o r . Bulky spin labels however, can produce considerable s t e r i c and ele c t r o n i c perturbations of the parent molecule. It is thus necessary to demonstrate that the spin labels bind to enzyme in the same fashion as a p a r t i c u l a r natural i n h i b i t o r (or a sub-strate) . - 25 -CHAPTER II THEORETICAL BACKGROUND Interpretation of this thesis i s based on equations developed from Bloch equations for the case where the popu-l a t i o n of one s i t e i s very much greater than that of the other s i t e . The r e l i a b i l i t y of the res u l t i s discussed in terms of the expected reasonable range of the transverse relaxation time for a methyl group on a macromolecule. The appropriate t h e o r e t i c a l background for the work i s thus explained in this chapter. A. THE LINESHAPE ANALYSIS FOR A MOLECULE UNDERGOING CHEMICAL  EXCHANGE WHEN THE POPULATION OF THE TWO SITES ARE GREATLY  DIFFERENT As mentioned in the introduction, the changes in NMR chemical s h i f t s and relaxation times can be used to obtain information on molecular interactions in solution. NMR spec-troscopy provides a powerful tool for the study of the i n t e r -action of i n h i b i t o r s with enzymes. The concentration of bound i n h i b i t o r s (those interacting with the enzyme), i s often limited however, due to the concentration of the enzyme. Subsequent observation of NMR spectra i s therefore d i f f i c u l t . Furthermore i n h i b i t o r s may undergo chemical exchange at a rate of the same order of magnitude as the NMR relaxation rates; t h i s slow time - 26 -s c a l e phenomenon bei n g a c h a r a c t e r i s t i c d i f f e r e n c e from o t h e r s p e c t r o s c o p i c methods. A l t h o u g h the s p e c t r a o f bound i n h i b i -t o r s cannot u s u a l l y be observed d i r e c t l y , q u a n t i t a t i v e i n f o r m a -t i o n can be deduced under c e r t a i n c o n d i t i o n s from the NMR spec-t r a o f f r e e i n h i b i t o r s . In o r d e r t o e x p l a i n the e f f e c t of c h e m i c a l exchange on the l i n e w i d t h of the NMR peak, c l a s s i c a l B l o c h e q u a t i o n s w i l l be d e v e l o p e d , and c o n d i t i o n s w i l l be de-f i n e d under which c e r t a i n i n f o r m a t i o n can be o b t a i n e d . Gutowsky and M c C a l l (80) used the c l a s s i c a l B l o c h equa-t i o n s t o q u a n t i t a t i v e l y r e l a t e n u c l e a r resonance l i n e s h a p e s w i t h the r a t e s o f f a s t , r e v e r s i b l e , c h e m i c a l exchange r e a c t i o n s . I t was l a t e r shown by M c C o n n e l l , t h a t the same r e l a t i o n s h i p c o u l d be more e a s i l y d e r i v e d u s i n g m o d i f i e d B l o c k e q u a t i o n s , t o i n c l u d e the e f f e c t s o f c h e m i c a l exchange ( 8 1 ) . The e x p e r i - ' m e n t a l r e s u l t s o f t h i s t h e s i s are i n t e r p r e t e d u s i n g e q u a t i o n s d e v e l o p e d i n the same manner f o r two s p i n systems, as those d e r i v e d by S w i f t and Connick (82) f o r a t h r e e s p i n system. C o n s i d e r a system where a r a p i d r e v e r s i b l e m o l e c u l a r p r o -c e s s t r a n s f e r s a n u c l e u s X back and f o r t h between two m o l e c u l a r environments A and B. U s i n g the B l o c k ' s n o t a t i o n , l e t u, v and M z denofae the components of the n u c l e a r m a g n e t i z a t i o n which are i n phase w i t h the component of the r f f i e l d r p t a t i n g a t a n g u l a r f r e q u e n c y t o , out o f phase w i t h t h i s r o t a t i n g r f f i e l d , and a l o n g t h e d i r e c t i o n o f the l a r g e s t a t i o n a r y f i e l d , r e s p e c t i v e l y . L e t M^ be the v a l u e o f M^ a t e q u i l i b r i u m . Then the B l o c k - 27 -equations in the frame rotating at frequency to, are M • - - & ( l b ) dM (M - M ) TTF " " % H 1 V - T, <lc' where w i s the nuclear resonance frequency (io0 = Y N H D ) » i n which and T 2 are the longitudinal and transverse relaxation times respectively. By introducing the notation M = u + iv (la) and (lb) reduce to §f + { ( W o - W ) i - ± } M = i W l M 0 (1) where M = M for a weak f i e l d H, at steady state, z o 1 J Now consider a chemical exchange. If the p r o b a b i l i t y of the nucleus jumping from s i t e A to B i s P A g 6 t , or vice versa P B A 6 t , and MA and Mg are the components of M at s i t e s A and B respectively, then the change in MA and Mg due to jumping can be written as dM, a = _ P M + P M St - " AB A BA B dM„ E = -P M + P M dt BA B + AB A - 28 -E q u a t i o n (2) can be w r i t t e n f o r M^ and M g t o i n c l u d e the c o n t r i b u t i o n from c h e m i c a l exchange dM „ St + + PAB " i A < V MA " P B A M B = i w l M o A <31> dM 1 „ ~dF + <T^ + PBA " 1 A ^ B ) M B - P A B M A = ^ l M o B <3b> where Aw A = O J A - OJ and AWg = coB - to. At the s t e a d y s t a t e , f^A = ^ B = 0 i n eq. (3a) and (3b). d t dt S o l v i n g the s i m u l t a n e o u s e q u a t i o n s f o r M A then y i e l d s < < T T 7 + P B A - i A V M o A - PBA MoB> 1 W l M B = P A B P B A " ( T T " + PAB " i A V ( T T 7 + PBA ' i A V However s i n c e A i s o v e r w h e l m i n g l y dominant t o B, then M Q A >> MoB* H e n c e P B A M O B C A N B E i 9 n o r e c 3 compared w i t h P B A M 0 ^ * T n e t o t a l s i g n a l M i s a l s o g i v e n , t o a v e r y good a p p r o x i m a t i o n , by . T h e r e f o r e M can be wr i t t e n as ( T 7 ~ + PBA - i A < V MoA J ; j ; P A B P B A - + PAB " l A w B } + PBA " l A V To d e r i v e an e x p r e s s i o n f o r v, the out o f phase component o f M, the i m a g i n a r y terms a re c l e a r e d from the denominator. From the r e s u l t i n g e q u a t i o n , the i m a g i n a r y terms are s e l e c t e d and r e a r r a n g e d t o g i v e - 29 -v = C- MoA" < T T - + PBA> 2 +( A w Bf l ) 2 The only assumption made u n t i l now i s that A i s in large excess over B. The numerator and the term in the f i r s t bracket are e s s e n t i a l l y frequency independent. The maximum value of v w i l l thus occur when the second term in the denominator i s zero, and the half-width at half-height for thi s Lorentzian-l i k e lineshape i s given to a good approximation, by the term in the f i r s t bracket in the denominator. The experimental half-width at half-height i s given by the symbol 1 . Under l i m i t i n g conditions, s i m p l i f i c a t i o n of equation 4 how-ever, leads to more useful equations. These are shown in Table I I - l together with the conditions of l i m i t i n g cases and the information obtainable. In the table A t o B = to - co B i s replaced by Ato = t o A - to B • In the slow exchange region, cu , can be replaced by to, since (4) - 3 0 -T a b l e 11-1 E x p e c t e d r e l a x a t i o n t i m e s i n p r e s e n c e o f t h e c h e m i c a l e x c h a n g e  when A i s t h e d o m i n e n t s p e c e i s i n s o l u t i o n s p e e d o f e x c h a n g e c o n d i t i o n o b s e r v a t i o n i n f o r m a t i o n o b t a i n a b l e T 2 s l o w L»%» ( } ) * , P ^ A < T > B » A W * - P B A 1 = 1 + f * p T 2 T 2 A f B P B A = 1 + P ( 4 - a ) P P BA , AB f a s t P B A W » T 2 B P B A 1 _ 1 + f B ^ T 2 T 2 A P B A f 4 - b ) A 0 J v e r y f a s t T " p B A > > ( T ) 2 ' A U F L  1 2 B D 1 2 B 1=1 + f l T 2 T 2 A B T 2 B C4-c) 1 K T ' D ' 2 B U T l s l o w T » P B A ' l B b A 1=1 + f P T l T 1 A P P r B A , AB f a s t T « P B A 1 I B B M 1=1 + f l T l T 1 A B T 1 B 1 K T ' D 1 1 B U f g i s t h e f r a c t i o n o f m o l e c u l e s on s i t e B a n d e x p r e s s e d a s P A B  P A B + P B A - 3 1 -o n l y a s i n g l e l a r g e peak due t o A would be observed w i t h the c o n d i t i o n QT] >> QBJ , and resonance f r e q u e n c y (to) w i l l be a l m o s t e q u a l t o t h a t o f pure A. T h i s i s a l s o t r u e i n the f a s t exchange r e g i o n s i n c e the f r e q u e n c y of the averaged peak would be c l o s e t o t h a t o f pure A a l s o due t o l a r g e e x c e s s o f pure A. When the o f f - r a t e ( d e f i n e d as P g A - the r a t e m o l e c u l e s change environment from B t o A ) * i s much slowe r than e i t h e r the r a t e o f r e l a x a t i o n or a change i n p r e c e s s i o n a l f r e q u e n c y , then the l i n e b r o a d e n i n g (~- - T^T1—) i s governed by the r a t e o f c h e m i c a l l 2 2A exchange. In the f a s t r e g i o n , the l i n e b r o a d e n i n g i s c o n t r o l l e d by both the o f f - r a t e and the change i n the p r e c e s s i o n a l f r e -quency, whereas i n the v e r y f a s t r e g i o n , c o n t r o l i s by the T 2 B r e l a x a t i o n p r o c e s s o n l y . The e q u a t i o n s shown i n Table I I - l f o r the l o n g i t u d i n a l r e -l a x a t i o n time o f observed peak are o b t a i n e d from e q u a t i o n l c i n a s i m i l a r manner as T 2, where the s o l e assumption i s t h a t s p e c i e s A i s much more c o n c e n t r a t e d than B i n the s o l u t i o n . As e x p e c t e d from e q u a t i o n l c , e q u a t i o n s f o r T^ are independent of the c h e m i c a l s h i f t d i f f e r e n c e i n the two s i t e s . However e q u a t i o n s f o r T^ a t both extreme l i m i t s o f exchange show the same forms as ones o b t a i n e d f o r T 0. * The t e r m i n o l o g y " o f f - r a t e " i s used s i n c e e q u a t i o n s de-v e l o p 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 e x p l a i n the i n t e r -a c t i o n between i n h i b i t o r s and enzymes. In t h i s s i t u a t i o n P B A d e s c r i b e s the r a t e o f r e l e a s e o f i n h i b i t o r s bound t o the enzyme. - 32 -B. EXPECTED LINEWIDTH IN NMR SPECTRA FOR A METHYL GROUP  ATTACHED TO A MACROMOLECULE One of the c r i t e r i a for choosing i n h i b i t o r s interacting with AchE i s that there should be present at least one methyl group to y i e l d a 3-fold increased signal to noise r a t i o . The protons of t h i s methyl group have i d e n t i c a l chemical s h i f t s and thus give spectra with no s p l i t t i n g s from scalar coupling. This gives another advantage since the expected magnetic re-laxation times T-^  and T 2 for a methyl group r i g i d l y attached to a spherical macromolecule were calculated by Werbelow and Marshall (83,84). They considered the relaxation for a three-spin system, including the ef f e c t of cross-correlation, and took the l i m i t that cross-correlation goes to zero. The r e l a -tionship of T 2 with the ro t a t i o n a l c o r r e l a t i o n time i s plotted in Figure I I - l , according to the following equation obtained from their c a l c u l a t i o n . i -3 4 V2 5T 2T T 6 1 T c 2 2 2 2' ( ' l 2 10r b c 1 + W T C 1 + 4co T C T i s the rot a t i o n a l correlation time in terms of the rotational c d i f f u s i o n constant for a sphere and r i s the proton-proton distance in a methyl group. The rot a t i o n a l c o r r e l a t i o n time in the magnetic resonance is expressed, for a spherical mole-cule, as - 33 -L o g [ l / T 2 ] 10 L o g[-cc0 F i g u r e 11-1 : A p l o t o f 1/ T 2 v s . r e c i p r o c a l 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 . A c u r v e i s o b t a i n e d f r o m E q u a t i o n (5), w h i c h c a l c u l a t e s 1/ T 2 f o r a m e t h y l g r o u p r i g i d l y a t t a c h e d t o a s p h e r i c a l m o l e c u l e w h o s 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 i s ~cc. - 34 -where R, n, k and T are the radius of the sphere, the solution v i s c o s i t y , the Boltzmann constant and the absolute temperature (85). Hence i f the molecular weight, the p a r t i a l s p e c i f i c volume of the molecule, and the solution v i s c o s i t y are known, the r o t a t i o n a l c o r r e l a t i o n time can be calculated, and thus the expected values for T 2. C. PLOTS TO OBTAIN BOUND LINEWIDTH There are two methods to deduce the bound NMR linewidth of an i n h i b i t o r from the linewidth actually observed when the rate of exchange i s in the very fast region. In both methods, i t i s assumed that the concentration of the i n h i b i t o r is much greater than the concentration of the enzyme. Starting from the same equation of T 2 for the very fast exchange region in Table I I - l ; 1 - 1 + U 1 T 2 T 2 A B T 2 B Gerig obtained the following expression ( 8 6 ) . ± - - L . = E ° • _L_ (7) T 2 T 2 A Io + % T 2 B In the above equation E Q and I Q are the i n i t i a l concentration of the enzyme and in h i b i t o r respectively, with K D being the d i s s o c i -ation constant representing for equilibrium E + I % C E I J 1 1 . EI. 7 p — and 7 = — now represent the linewidths of nuclei in free and bound i n h i b i t o r s . - 35 -Kato made another assumption; namely t h a t the l i n e w i d t h of the bound i n h i b i t o r i s much g r e a t e r than t h a t of the f r e e i n h i b i t o r , and thus o b t a i n e d the f o l l o w i n g e q u a t i o n ( 7 3 ) . 1 Top, = E o V ~ KD <8> T2 " ^2A For both e q u a t i o n s , the bound l i n e w i d t h i s o b t a i n e d as the g r a d i e n t o f a s t r a i g h t l i n e . A l t h o u g h i t was n e c e s s a r y t o make two assumptions t o o b t a i n e q u a t i o n 7, t h i s e q u a t i o n has an advantage t h a t the known v a l u e of i s not r e q u i r e d t o o b t a i n the v a l u e o f —-. F u r t h e r m o r e , i t i s p o s s i b l e t o o b t a i n the K i2 D v a l u e as the y i n t e r c e p t o f a s t r a i g h t l i n e o b t a i n e d from equa-t i o n 7. The K D v a l u e o b t a i n e d i n t h i s manner i s not a c c u r a t e however, as g e n e r a l l y K D << I Q . These two e q u a t i o n s are used i n Chapter IV t o p l o t the r e s u l t s and c a l c u l a t e the bound l i n e w i d t h ^ T2B - 36 -CHAPTER I I I EXPERIMENTAL METHODS A. PURIFICATION OF ACETYLCHOLINESTERASE i ) E x t r a c t i o n o f AchE from E l e c t r i c E e l i n High S a l t Media L i v e e l e c t r i c e e l s were k i l l e d by p a c k i n g them i n t o a c r u s h e d i c e - w a t e r m i x t u r e f o r h hour and the main e l e c t r i c organs were removed by d i s s e c t i o n , y i e l d i n g 400-800 g per e e l . 3 F r e s h e l e c t r i c t i s s u e was c u t up i n t o a p p r o x i m a t e l y 2-cm cubes. E l e c t r i c t i s s u e p i e c e s n o t used i m m e d i a t e l y were f r o z e n i n l i q u i d n i t r o g e n and s t o r e d at d r y i c e temperature u n t i l needed. The f o l l o w i n g p rocedure was c a r r i e d out a t 4°C t o a v o i d a u t o l y s i s (27,40,87). P i e c e s o f f r e s h or f r o z e n t i s s u e were homogenized i n one volume o f 5% s u c r o s e s o l u t i o n i n a Waring B l e n d e r f o r 30 seconds a t low s e t t i n g . The homogenate was then c e n t r i f u g e d f o r h hour a t 10,000 rpm i n the S o r v a l RC2B r e -f r i g e r a t e d c e n t r i f u g e and s u p e r n a t a n t l i q u i d c o n t a i n i n g 10-20% of the t o t a l a c t i v i t y was poured o f f . The r e s i d u e was rehomo-g e n i z e d w i t h two volumes o f 5% s u c r o s e s o l u t i o n and c e n t r i f u g e d as b e f o r e . T h i s r e s i d u e was then homogenized i n a 3-5 f o l d volume of h i g h s a l t b u f f e r (2 M N a C l , 20 mM sodium phosphate, 2 mM Na 2EDTA pH 7.0 a d j u s t e d a t room temperature) f o r 30 seconds and c e n t r i f u g e d a t 31,000 rpm f o r 6 hours i n the Beckman - 37 -L3-50 r e f r i g e r a t e d p r e p a r a t i v e u l t r a c e n t r i f u g e . The s u p e r -n a t a n t was r e t a i n e d and the h i g h s a l t e x t r a c t i o n was r e p e a t e d w i t h the r e s i d u e . S u p e r n a t a n t s from both e x t r a c t i o n s , which accounted f o r 60-70% of the t o t a l AchE a c t i v i t y i n the o r i g i n a l homogenate, were combined and f i l t e r e d t hrough g l a s s w o o l . The f i l t r a t e was then d i l u t e d w i t h an e q u a l volume of no s a l t b u f -f e r (20 mM sodium phosphate, pH 7.0) t o g i v e the f i n a l e x t r a c t of 1 M N a C l , as r e q u i r e d f o r the e n s u i n g a f f i n i t y chromatography. Some o f the e x t r a c t s were p r e p a r e d by l e t t i n g the homo-genate s t a n d o v e r n i g h t i n h i g h s a l t b u f f e r i n the r e f r i g e r a t o r p r i o r t o c e n t r i f u g a t i o n . In t h i s c a s e , 90% o f the AchE was r e -c o v e r e d i n the f i r s t e x t r a c t i o n and f u r t h e r e x t r a c t i o n was o m i t t e d . T h i s d e l a y i n the e x t r a c t i o n d i d not seem t o a l t e r the form o f AchE and d i d not a f f e c t the r e c o v e r y and the p u r i -f i c a t i o n o f AchE by a f f i n i t y columns. i i ) P u r i f i c a t i o n o f AchE by A f f i n i t y Chromatography a) P r e p a r a t i o n o f the column The N - m e t h y l a c r i d i n i u m c o u p l e d Sepharose 2B a f f i n i t y m a t r i x used i n a f f i n i t y chromatography had been p r e p a r e d by Webb a c c o r d i n g t o p u b l i s h e d methods (1 8 ) . The procedure i n -v o l v e s the attachment o f s p e c i f i c , r e v e r s i b l e AchE i n h i b i t o r s t o a Sepharose r e s i n m a t r i x by means of the c o u p l i n g agent, cyanogen bromide. When a crude e x t r a c t o f AchE i s a p p l i e d t o the chromatography column c o n t a i n i n g the m o d i f i e d r e s i n , the AchE i s s e l e c t i v e l y r e t a i n e d and the enzyme may then be e l u t e d - 38 -from the column w i t h a s a l t g r a d i e n t or a s o l u t i o n c o n t a i n i n g a r e v e r s i b l e i n h i b i t o r w i t h a s t r o n g e r b i n d i n g c o n s t a n t than the l i q u i d a t t a c h e d t o the m a t r i x . The a f f i n i t y m a t r i x used i n t h i s case had a N - m e t h y l a c r i d i n i u m l i g a n d c o n c e n t r a t i o n of ~ 0.4 ymol/mfi, packed g e l , which g i v e s the most e f f i c i e n t r e c o v e r y and a good s p e c i f i c a c t i v i t y (27). The m o d i f i e d Sepharose 2B g e l was packed i n 10 ml d i s -p o s a b l e s y r i n g e s w i t h f i t t e d bottom d i s c s made from porous p o l y p r o p y l e n e and e q u i l i b r a t e d w i t h chromatography b u f f e r (20 mM sodium phosphate, IM N a C l , pH 7.0) by f l u s h i n g the packed column t h o r o u g h l y w i t h the b u f f e r . A f t e r each use the column was washed w i t h a 5M G u a n i d i n e -HC1 s o l u t i o n i n chromatography b u f f e r . One column volume of G u a n i d i n e - H C l s o l u t i o n was passed through the column and the f l o w was stopped t o l e t the g e l s t a n d i n the s o l u t i o n f o r a minimum of 12 h o u r s . A f t e r t h o r o u g h l y washing w i t h chromato-graphy b u f f e r ( a p p r o x i m a t e l y f i v e - f o l d volume) the column was ready f o r r e l o a d i n g . The performance of the column d e t e r i o r a t e d however a f t e r r e p e a t e d use, due t o b u i l d - u p of m a t e r i a l on top of the g e l and growth of b a c t e r i a . T h e r e f o r e i t was n e c e s s a r y t o t r e a t the column p a c k i n g w i t h t r y p s i n a f t e r a c e r t a i n number of r u n s . T h i s was done by suspending the g e l i n t r y p s i n b u f f e r (0.5 M N a C l , 5 mM T r i s HC1, pH 7.5) and adding f r e e z e d r i e d t r y p s i n t o a f i n a l c o n c e n t r a t i o n o f 0.04 mg/m£. Then the s o l u t i o n was - 39 -i n c u b a t e d a t room temperature f o r a few days. The r e g e n e r a t e d g e l was washed and e q u i l i b r a t e d w i t h chromatography b u f f e r be-f o r e use and gave j u s t as good a performance as newly p r e p a r e d g e l . b) Running o f the column The A c h E - c o n t a i n i n g e x t r a c t s , p r e p a r e d as d e s c r i b e d i n S e c t i o n _ i , were l o a d e d onto the chromatography columns a t a f l o w of l e s s than one column volume per hour. A f t e r l o a d i n g was co m p l e t e d , the non-adsorbed p r o t e i n s were washed o f f w i t h chromatography b u f f e r a t the same f l o w r a t e u n t i l the &280 r e a d i n g was 0.3 or l e s s ( a p p r o x i m a t e l y f i v e v o l u m e s ) . AchE was e l u t e d w i t h a p p r o x i m a t e l y h a l f a column volume o f 20 mM Decamethonium Bromide i n chromatography b u f f e r at a f l o w r a t e o f 0.1 column volumes per hour and was c o l l e c t e d i n f r a c t i o n s o f a p p r o x i m a t e l y 1 ml. The e l u t i o n was completed w i t h chroma-togr a p h y b u f f e r a t the same f l o w r a t e . A l l o p e r a t i o n s were done a t 5°C. The absorbance a t 280 nm o f each f r a c t i o n was determined and the e l u t i o n p r o f i l e was p l o t t e d . (A t y p i c a l e l u t i o n p r o -f i l e i s g i v e n i n F i g u r e I I I - l . ) The a c t i v i t y of AchE was measured f o r the f r a c t i o n s around the major peak and the spe-c i f i c a c t i v i t i e s were c a l c u l a t e d . The f r a c t i o n s w i t h low s p e c i f i c a c t i v i t i e s were combined and d i a l i s e d at 5°C t h r e e times a g a i n s t b u f f e r f o r 24 hours eac h , i n o r d e r t o remove the decamethonium bromide. The - 40 -F i g u r e 111-1: An e l u t i o n p r o f i l e o f A c h E f r o m an a f f i n i t y c o l u m n . - 41 -r e m a i n i n g s o l u t i o n was then d i l u t e d t e n times and r e p u r i f i e d by a f f i n i t y chromatography as d e s c r i b e d above. The r e s u l t i n g AchE s o l u t i o n was combined w i t h the f r a c t i o n s o f h i g h s p e c i f i c a c t i v i t y o f the o r i g i n a l p u r i f i c a t i o n , g i v i n g an a c t i v i t y o f a p p r o x i m a t e l y 9 mM o f a c e t y l t h i o c h o l i n e h y d r o l i z e d per mg of p r o t e i n per min. i i i ) A c e t y l c h o l i n e s t e r a s e Assay A l l a c t i v i t i e s f o r AchE i n t h i s t h e s i s were assayed by the p h o t o m e t r i c method developed by E l l m a n e t a l (88), u s i n g the s u b s t r a t e analogue a c e t y l t h i o c h o l i n e . The p r i n c i p l e of the method i s the measurement o f the r a t e of p r o d u c t i o n o f t h i o -c h o l i n e as a c e t y l t h i o c h o l i n e i s h y d r o l y z e d . T h i s i s accom-p l i s h e d by o b s e r v i n g the e n s u i n g r e a c t i o n o f the t h i o l a t e a n i o n w i t h 5 , 5 1 - d i t h i o b i s - 2 - n i t r o b e n z o i c a c i d (DTNB), which produces the y e l l o w a n i o n o f 5 - t h i o - 2 - n i t r o - b e n z o i c a c i d . The r a t e o f c o l o u r f o r m a t i o n i s a d i r e c t measure o f the r a t e o f a c e t y l t h i o -c h o l i n e h y d r o l y s i s , as the r e a c t i o n w i t h DTNB i s s u f f i c i e n t l y f a s t compared t o the a c e t y l t h i o c h o l i n e h y d r o l y s i s . The a ssay m i x t u r e was p r e p a r e d by p i p e t t i n g 3.0 ml o f 0.1 M sodium phosphate pH 8.0, 25 u£ o f 0.075 M a c e t y l t h i o c h o -l i n e , 100 \il o f 0.01 M DTNB i n 0.1 M sodium phosphate pH 7.0 and 50 \il o f enzyme s o l u t i o n i n 0.1 - 1 M NaCl chromatography b u f f e r i n t o a 1 cm c u v e t t e and m i x i n g i t t h o r o u g h l y . The i n -c r e a s e i n absorbance a t 412 nm was measured i n a Z e i s s PMQII s p e c t r o m e t e r a t room t e m p e r a t u r e . AchE s o l u t i o n s were d i l u t e d - 42 -to ensure that there was enough substrate to maintain a linear rate of hydrolysis for at least 2 minutes. Enzyme a c t i v i t i e s are reported in units, where each unit of en-zyme w i l l change the absorbance at 412 nm by 0 .1 ml min ^. Spe-c i f i c a c t i v i t y i s calculated in mmole of acetylcholine hydro-lyzed per mg of protein per min, where the protein concentra-tion (mg/m£) for the AchE was determined using the published 1% value £280nm = -^ .O (13) and the rate of hydrolysis of a c e t y l -thiocholine was calculated using the extinction c o e f f i c i e n t for 4 -1 -1 the t h i o l a t e anion, e^^nm = 1*36 x 10 M cm at pH 8.0. B. CONVERSION OF AchE TO US FORM i) Trypsin Digestion AchE solutions obtained as above were treated with trypsin to form the globular l i s form. The AchE solution was dialysed against 100 volumes of trypsin buffer at 4°C twice for 24 hrs. Then trypsin dissolved in the same buffer was added to the AchE solution to a f i n a l concentration of 1 mg Trypsin per 25 ml AchE solution approx. 0.7). The solution was incubated at room temperature for 20 minutes. The reaction was terminated by adding soy bean trypsin i n h i b i t o r (STI), where STI had a concentration of 2 mg per 25 ml of AchE solution. (The a c t i v i t y of AchE was measured before and after the digestion and i t was found that neither trypsin digestion nor the presence of STI changed the a c t i v i t y of AchE). - 4 3 -The solution was further p u r i f i e d by a f f i n i t y chromato-graphy. To thi s end i t was dil u t e d to 400 units of a c t i v i t y per ml with 0.1 M NaCl chromatography buffer and then loaded onto the equilibrated column and washed and eluted with 20 mM decamethonium bromide as described in Section A - i i - b , however 0.1 NaCl chromatography buffer was used instead of regular IM NaCl chromatography buffer throughout. Under these conditions the 11S form of AchE i s s e l e c t i v e l y p u r i f i e d (27). However for la t e r batches, this step was omitted since i t was found that incubating o r i g i n a l AchE solutions with trypsin for a somewhat longer period of time (approx. 1 hour) leads to conversion into the 11S form in almost 100% y i e l d . No further conversion or loss of a c t i v i t y could be detected. i i ) Characterization of Converted AchE For each preparation of trypsin treated AchE solution, the extent of conversion to the 11S form of AchE was tested by is o k i n e t i c sedimentation in a sucrose gradient which i s ar-ranged so that macromolecules of d i f f e r e n t molecular weight sediment at constant rates, respectively. The required expo-nentional sucrose gradient, whose range had been calculated previously (87), was prepared by pumping a 29.3% sucrose solution in chromatography buffer into a 10% sucrose solution in the same buffer, which in turn was pumped at exactly the same speed (use of a p e r i s t a l t i c pump) into the gradient tubes (7.3 cm c e l l u l o s e n i t r a t e tubes for Beckman SW41 swinging - 44 -bucket r o t o r ) . P r e p a r e d g r a d i e n t s were c o o l e d t o 4°C and a t h i n l a y e r o f AchE sample ( a l s o p r e c o o l e d ) was c a r e f u l l y a p p l i e d o n t o the t o p o f the s u c r o s e g r a d i e n t . Each sample c o n t a i n e d 100 u& of AchE s o l u t i o n c o n t a i n i n g 300-1000 u n i t s o f a c t i v i t y and 100 y£ o f s t a n d a r d s ; f 3 - g a l a c t o s i d a s e and c a t a -l a s e i n 10% s u c r o s e s o l u t i o n . The s e d i m e n t a t i o n c o n s t a n t s o f these p r o t e i n s a re 15.9S and 11.3S, r e s p e c t i v e l y (28,29). The g r a d i e n t s were c e n t r i f u g e d a t 4°C i n a Beckman 23-50 u l t r a c e n -t r i f u g e f o r 19-20 h r s a t 40,000 rpm. F o l l o w i n g the c e n t r i f u g a -t i o n the g r a d i e n t s were f r a c t i o n a t e d by c a r e f u l l y i n s e r t i n g a c a p i l l a r y tube down onto the bottom o f the tube and s l o w l y emptying i t s c o n t e n t s , c o l l e c t i n g f r a c t i o n s o f a p p r o x i m a t e l y 0.5 ml volume. AchE was d e t e c t e d by i t s a c t i v i t y , w h i l e the c a t a l a s e was l o c a t e d by the c h a r a c t e r i s t i c absorbance a t 405 nm. B - g a l a c t o s i d a s e was d e t e c t e d by a s p e c i f i c a c t i v i t y assay ( 8 ) . A c t i v i t i e s o f AchE i n s u c r o s e g r a d i e n t , which was taken b e f o r e and a f t e r the t r e a t m e n t w i t h t r y p s i n , a re p l o t t e d a g a i n s t f r a c t i o n number i n F i g u r e I I I - 2 . I t i s c l e a r from the f i g u r e and d a t a from o t h e r r e p o r t s (27,40) t h a t n e a r l y 100% o f AchE i s c o n v e r t e d t o the 11S form. The 11S AchE was c o n c e n t r a t e d i n t o D 20 b u f f e r as d e s c r i b e d i n the f o l l o w i n g s e c t i o n , u s u a l l y w i t h o u t d e l a y . However, when the enzyme c o u l d n ot be used i m m e d i a t e l y , i t was s t o r e d (up t o one month) i n 4 M NaCl b u f f e r (20 mM sodium phosphate, pH 7.0). - 45 -F i g u r e I I I - 2 : S u c r o s e g r a d i e n t p r o f i l e o f A c h E b e f o r e ( a ) a n d a f t e r ( b ) t r e a t m e n t w i t h t r y p s i n . - 46 -C. PREPARATION OF AchE SAMPLES IN D 20 BUFFER i ) C o n c e n t r a t i o n o f the Enzyme w i t h the Amicon F i l t e r 11S AchE (10 mil : s o l u t i o n , see above s e c t i o n ) was f i l l e d i n t o an Amicon D i a f l o U l t r a f i l t e r a t i o n a p p a r a t u s (equipped w i t h an UM10 f i l t e r ) , which had been e q u i l i b r a t e d p r e v i o u s l y w i t h chromatography b u f f e r . The s o l u t i o n was c o n c e n t r a t e d t o almost n i l volume by a p p l y i n g a p r e s s u r e of 14-20 p s i . (UM10 f i l t e r r e t a i n s macromolecules w i t h a m o l e c u l a r weight > 10,000.) Then the a p p a r a t u s was topped up w i t h a p p r o x i m a t e l y 10 ml o f D 20 b u f f e r (0.1 M N a C l , 20 mM sodium phosphate, i n D 20, pH 7.0; a c t u a l meter r e a d i n g , not c o r r e c t e d f o r d e u t e r i u m i s o t o p e e f -f e c t ) and the s o l u t i o n a g a i n c o n c e n t r a t e d t o a l m o s t n i l volume. T h i s p r o c e s s was r e p e a t e d once more and then D 20 b u f f e r was added t o a f i n a l A 2 g ^ r e a d i n g of around 3.5. T h i s c o r r e s p o n d s t o 18 uM o f 80,000 c a t a l y t i c s u b u n i t s of AchE, a f t e r t a k i n g into account the c o n t r i b u t i o n s from t r y p s i n and STI. By t h i s method 65-75% of the enzyme was r e c o v e r e d a f t e r c o n c e n t r a t i o n i n t o D 20 b u f f e r , w h i l e t h e r e was no a p p r e c i a b l e l o s s i n s p e c i f i c a c t i v i t y . T h i s i n d i c a t e s t h a t a l l the enzyme r e c o v e r e d i s c a t a l y t i c a l l y a c t i v e and p r o b a b l y has not changed i t s form. The c o n c e n t r a t e d enzyme sample i n D 20 b u f f e r was s t o r e d i n a p l a s t i c v i a l a t 4°C and used f o r NMR e x p e r i m e n t s w i t h i n two weeks. - 47 -i i ) Other Methods f o r C o n c e n t r a t i n g AchE i n t o D.,0 B u f f e r The f r e e z e d r y i n g method i s commonly used t o c o n c e n t r a t e enzymes i n t o D 20 f o r NMR s t u d i e s . AchE s o l u t i o n s were passed through sephadex G25 column t o remove decamethonium bromide and then f r e e z e d r i e d t w i c e under v a r i o u s c o n d i t i o n s by chang-i n g the f r e e z i n g t e m p e r a t u r e , f r e e z i n g media and f r e e z e d r y i n g t i m e . However i t was found t h a t the enzyme was not s t a b l e enough, e s p e c i a l l y a g a i n s t f r e e z i n g , and even under the most f a v o r a b l e c o n d i t i o n s , when the sample was f r o z e n i n 5% g l y c e r o l s o l u t i o n a t l i q u i d N 2 t e m p e r a t u r e , o n l y 35% o f o r i g i n a l enzyme a c t i v i t y c o u l d be r e c o v e r e d . The l o s s o f a c t i v i t y of enzyme was a t t r i b u t e d t o the d e n a t u r a t i o n d u r i n g the p r o c e s s , s i n c e no l o s s o f p r o t e i n c o u l d be o b s e r v e d . Another method of c o n c e n t r a t i n g the enzyme i n t o D 20 b u f f e r t a k e s advantage of the g r e a t e r s p e c i f i c g r a v i t y o f D 20. A c o n c e n t r a t e d sample of the enzyme i n aqueous (H 20) s o l u t i o n was p l a c e d on t o p o f a l a r g e amount of D 20 b u f f e r by c a r e f u l l y i n j e c t i n g D 20 b u f f e r onto the bottom of the tube c o n t a i n i n g the aqueous sample. A f t e r c e n t r i f u g a t i o n a t 40,000 rpm the s o l u -t i o n was f r a c t i o n a t e d i n the same manner as d e s c r i b e d i n Sec-t i o n B - i i . A s m a l l amount of t r i t i u m was added t o the aqueous l a y e r t o m onitor the e x t e n t o f H 20 m i x i n g w i t h D 20 d u r i n g the c e n t r i f u g a t i o n . The r a d i o a c t i v i t y of t r i t i u m i n each f r a c t i o n was counted u s i n g a N u c l e a r C h i c a g o Mark V l i q u i d s c i n t i l l a t i o n c o u n t e r . I t was p o s s i b l e t o c o n c e n t r a t e the enzyme i n t o the - 48 -D 20 b u f f e r w i t h o u t d e n a t u r a t i o n or m i x i n g o f the D 20 and H 20 b u f f e r s o l u t i o n s . (A t y p i c a l p r o f i l e of enzyme a c t i v i t y d i s -t r i b u t i o n i n a tube i s shown i n F i g u r e I I I - 3 . ) However i t proved d i f f i c u l t t o s t o p the c e n t r i f u g e e x a c t l y a t the moment when the enzyme had been c o n c e n t r a t e d w e l l enough at the bottom o f the t u b e , but had not y e t been p r e s s e d a g a i n s t the bottom of the tube y e t , where i t would g e t p e l l e t i z e d and s t i c k t o the tube. Changing of the d e n s i t y g r a d i e n t a t the bottom of the tube w i t h s a l t s , s u c r o s e e t c . i n o r d e r t o slow the sedimenta-t i o n down d i d not s o l v e the problem. Moreover the presence of a c o n v e c t i o n c u r r e n t made t h i s method r a t h e r u n r e l i a b l e . D. PREPARATION OF MODIFIED ENZYME AND ITS VERIFICATION i ) P r e p a r a t i o n o f E s e r o l i n e E s e r o l i n e , the h y d r o l y s i s p r o d u c t o f e s e r i n e was p r e p a r e d i n a manner s i m i l a r t o the method used by E l l i s ( 9 0 ) . E s e r i n e s u l f a t e (0.5 g) was h y d r o l y z e d by s t i r r i n g i t f o r 6 hours at room temperature i n a 10% NaOH s o l u t i o n (5 mS,) under a n i t r o g e n atmosphere. The aqueous r e a c t i o n m i x t u r e was c o n t i n u o u s l y e x t r a c t e d w i t h e t h e r f o r 12 h o u r s . The o r g a n i c l a y e r was d r i e d over MgSO^ and f i l t e r e d under a N 2 stream. E v a p o r a t i o n o f the e t h e r gave the crude e s e r o l i n e , which was r e c r y s t a l l i z e d from a m i x t u r e o f benzene and p e t r o l e u m e t h e r and washed w i t h p e t r o -leum e t h e r . A l l s o l v e n t s were purged w i t h n i t r o g e n b e f o r e use and the p r o d u c t was s t o r e d under n i t r o g e n i n the f r e e z e r . m.p. = 113-115°C, MS: M + = 218 - 49 -10 20 30 F r a c t i o n Number F i g u r e 1 1 1 - 3 : An enzyme d i s t r i b u t i o n i n a t u b e a f t e r c e n t r i f u g a t i o n . A c t i v i t i e s o f A c h E a n d r a d i o a c t i v e c o u n t s o f t r i t i u m , w h i c h was o r i g i n a l l y m i x e d w i t h H^O s o l u t i o n o f A c h E , a r e p l o t t e d a g a i n s t t h e p o s i t i o n o f a c e n t r i f u g a t i o n t u b e . The s m a l l number o f f r a c t i o n c o r r e s p o n d s t o t h e b o t t o m o f a t u b e . I t i n d i c a t e s A c h E i s c o n c e n t r a t e d a t t h e b o t t o m o f t h e c e n t r i f u g a t i o n t u b e w i t h o u t s i g n i f i c a t amount o f H^O m i x i n g i n t o t h e D 9 0 l a y e r . - 50 -i i ) S u l f o n y l a t i o n o f AchE M e t h a n e s u l f o n y l f l u o r i d e was used f o r the s u l f o n y l a t i o n of AchE, s i n c e i t i s an i r r e v e r s i b l e i n h i b i t o r and produces an enzyme which i s s u l f o n y l a t e d e x c l u s i v e l y a t the e s t e r a t i c s i t e (76,91,92,93). The more r e a d i l y a v a i l a b l e s u l f o n y l c h l o r i d e i s t o o r e a c t i v e i n b a s e - c a t a l y z e d h y d r o l y s i s and l e a d s t o n o n s p e c i -f i c s u l f o n y l a t i o n of r e a c t i v e groups i n p r o t e i n s ( 9 4 ) . A l s o m e t h a n e s u l f o n y l AchE can be c o m p l e t e l y r e a c t i v a t e d , i n d i c a t i n g the enzyme does not go t h r ough f u r t h e r m o d i f i c a t i o n . M e t h a n e s u l f o n y l f l u o r i d e was p r e p a r e d as f o l l o w s : a m i x t u r e o f 10 g of MeSC^Cl and 30 g o f 70% aqueous KF s o l u t i o n was d i s t i l l e d and the f r a c t i o n b o i l i n g a t 124-125°C was c o l -l e c t e d . S i n c e the IR-spectrum s t i l l i n d i c a t e d the presence of s m a l l amounts of s u l f o n y l c h l o r i d e , the o r g a n i c l a y e r was t h o r o u g h l y washed w i t h c o l d water i n o r d e r t o h y d r o l y z e r e s i d u a l s t a r t i n g m a t e r i a l . R e d i s t i l l a t i o n gave pure m e t h a n e s u l f o n y l f l u o r i d e (bp 125°C, no more IR a b s o r p t i o n s at 1165 and 965 cm , MS : M+ = 98) . A s t o c k s o l u t i o n o f MeSC^F was p r e p a r e d by d i s s o l v i n g 200 yJi of Me'SC^F i n 5 ml of i s o p r o p a n o l ( i s o p r o p a n o l i s known t o be the l e a s t damaging t o the enzyme and i t was found t h a t the a d d i t i o n o f i s o p r o p a n o l (up t o 2%) t o the enzyme s o l u t i o n d i d not d e c r e a s e the a c t i v i t y ) . A s m a l l a l i q u o t of t h i s s o l u t i o n was added t o the s o l u t i o n of 11S AchE and a f t e r two hours a second a l i q u o t , amounting t o an approx. 500 f o l d molar e x c e s s - 51 -o f MeSG^F over AchE. The m i x t u r e was kept a t room temperature and the a c t i v i t i e s were m o n i t o r e d . A f t e r 6 h r s the enzyme s o l u t i o n r e t a i n e d 1/1000 o f i t s o r i g i n a l a c t i v i t y and the i n -h i b i t e d enzyme d i d not r e c o v e r i t s a c t i v i t y f o r a t l e a s t f o u r days. The m o d i f i e d enzyme was c o n c e n t r a t e d i n t o D 20 b u f f e r w i t h the Amicon f i l t e r as d e s c r i b e d i n S e c t i o n C - i . E x c e s s MeS0 2F and a l c o h o l were a l s o removed d u r i n g t h i s p r o c e s s . The p r e -pared s o l u t i o n s were used f o r NMR e x p e r i m e n t s w i t h i n a few days. E. STABILITY OF THE ENZYME In o r d e r t o a s s u r e r e l i a b i l i t y of the o b t a i n e d r e s u l t s , the s t a b i l i t y o f the enzyme towards the a p p l i e d s t o r a g e and e x p e r i m e n t a l c o n d i t i o n s was checked. 1) A f t e r s t o r a g e f o r one month i n 4 M NaCl b u f f e r at 4°C, 11S AchE s o l u t i o n was d i l u t e d i n t o s t a n d a r d 1 M NaCl chromatography b u f f e r and a c t i v i t y and u n i f o r m i t y o f the en-zyme were checked. ( U n i f o r m i t y was checked by i s o k i n e t i c s u c r o s e g r a d i e n t s e d i m e n t a t i o n as d e s c r i b e d i n S e c t i o n B - i i . ) 2) I t i s known t h a t AchE tends t o form a g g r e g a t e s i n low s a l t media (13,21) even i n a form which does not c o n t a i n a t a i l ( 9 5 ) , and i t was t h e r e f o r e t e s t e d whether t h i s o c c u r r e d i n the 0.1 M N a C l - D 2 0 - b u f f e r used f o r the NMR measurements. An a u t h e n t i c sample ( c a . 18 umole AchE i n D o0 b u f f e r ) was - 52 -t e s t e d f o r u n i f o r m i t y by i s o k i n e t i c s u c r o s e g r a d i e n t sedimen-t a t i o n a f t e r exposure t o e x p e r i m e n t a l c o n d i t i o n s . (Sedimenta-t i o n t e s t as i n S e c t i o n B - i i ; g r a d i e n t was prepare d w i t h 0.1 M NaCl b u f f e r i n s t e a d o f 1 M NaCl b u f f e r , however, and the AchE s o l u t i o n was loaded w i t h o u t d i l u t i o n . ) 3) To t e s t the s t a b i l i t y o f the enzyme under experimen-t a l c o n d i t i o n s an NMR tube was l e f t i n the machine f o r 24 h r s . S m a l l a l i q u o t s were taken p e r i o d i c a l l y and checked f o r t h e i r a c t i v i t y . F. NMR MEASUREMENTS A l l NMR s p e c t r a were r e c o r d e d on a V a r i a n XL100 NMR sp e c t r o m e t e r and/or a 270 MHz PMR-FT s p e c t r o m e t e r . 300 u£ ( f o r XL100) or 500 y£ ( f o r 270 MHz s p e c t r o m e t e r ) of the l is AchE s o l u t i o n were mixed w i t h a s m a l l volume (~35u£) of D 00 b u f f e r , c o n t a i n i n g the d e s i r e d amount o f i n h i b i t o r and sodium 3 - t r i m e t h y l s i l y l p r o p i o n a t e - 2 , 2 , 3 , 3 - d ^ (TSP) and f i l l e d i n t o 5 mm NMR t u b e s . A f t e r i n s e r t i o n o f the samples i n t o the NMR probe i t was n e c e s s a r y t o w a i t 30-45 minutes u n t i l the temper-a t u r e was e q u i l i b r a t e d . T h i s was assumed t o be the case when AV was l e s s than 0.1 Hz over a p e r i o d of f i v e minutes f o r the p o s i t i o n o f the TSP peak. Then the homogeneity o f the magnet was a d j u s t e d so t h a t the w i d t h o f the TSP peak was l e s s than 0.8 Hz. P o s i t i o n and peak w i d t h o f the TSP s i g n a l were a g a i n checked a f t e r each measurement t o ensure the s t a b i l i t y o f the - 53 -magnetic f i e l d d u r i n g the a c q u i s i t i o n o f the s i g n a l . Whenever e i t h e r a s h i f t i n TSP peak p o s i t i o n o f more than 0.2 Hz or a b r o a d e n i n g o f the TSP peak w i d t h o f more than 0.2 Hz was found, the spectrum was d i s c a r d e d . To change the c o n c e n t r a t i o n o f an i n h i b i t o r , a c e r t a i n amount of a s t o c k s o l u t i o n was added d i r e c t l y i n t o the NMR tube. Measurements were resumed a f t e r temperature e q u i l i b r i u m and r e a d j u s t m e n t o f the homogeneity of the magnet. S p e c t r a f o r a l l i n h i b i t o r s i n neat D 20 b u f f e r were r e c o r d e d s e p a r a t e l y . ( C o n c e n t r a t i o n s as i n e n z y m e - i n h i b i t o r e x p e r i m e n t s . ) A r e l a t i v e l y l a r g e peak from r e s i d u a l w a t e r , which appeared i n the v i c i n i t y o f the peak of i n t e r e s t , c o u l d be d e c r e a s e d i n a m p l i t u d e by use o f a band r e j e c t f i l t e r and a 100 Hz low pass f i l t e r ( 96). Two f i l t e r s were connected i n s e r i e s t o the o u t -l e t o f the XL100 e x t e r n a l f i l t e r and c o u l d be used whenever the s p e c t r a l w i d t h was l e s s than 100 Hz. T h i s had the e f f e c t o f i n c r e a s i n g the dynamic range o f the XL100 computer memory c a p a c i t y . ( F i l t e r s were p r e p a r e d by the d e p a r t m e n t a l e l e c t r o n i c shop.) T y p i c a l NMR parameters were ( F i g u r e s i n b r a c k e t s r e f e r t o 270 MHz s p e c t r o m e t e r ) : Spectrum w i d t h = 100-130 Hz (200 H z ) , A c q u i s i t i o n time = 5 sec (5.12 s e c ) , P u l s e d e l a y = 0 (393.22 msec), P u l s e w i d t h = 25 usee (6.50 u s e e ) , S e n s i t i v i t y enhancement = 3 sec (none), S p i n -n i n g r a t e = 35-40 c p s , Temperature = 17-30°C (io°C), Number of t r a n s i t i o n s = 300-1000 s c a n s . - 54 -CHAPTER IV RESULTS A. STABILITY AND PURITY OF THE ENZYME As r e p o r t e d i n S e c t i o n I I I - A - i i - b , t h e p u r i f i e d enzyme had a s p e c i f i c a c t i v i t y o f a p p r o x i m a t e l y 9 mM o f a c e t y l t h i o c h o l i n e h y d r o l i z e d p e r mg o f p r o t e i n p e r m i n . S i n c e the amount o f AchE w h i c h h y d r o l y z e s 1 ymole o f a c e t y l c h o l i n e p e r min would h y d r o l i z e 0.87 ymoles o f a c e t y l t h i o c h o l i n e p e r min (2 3 , 2 4 ) , t h e above s p e c i f i c a c t i v i t y c o r r e s p o n d s t o 10.34 mmoles o f a c e t y l -c h o l i n e h y d r o l y z e d / m g min. C u r r e n t l y , the maximum r e p o r t e d r e p r o d u c i b l e s p e c i f i c a c t i v i t y o f AchE i s 10.8 mmoles o f a c e t y l -c h o l i n e h y d r o l y z e d / m i n , mg ( 1 3 , 1 8 , 9 6 ) . S i n c e 11S AchE w i t h t h i s s p e c i f i c a c t i v i t y shows s i n g l e f o r m by e l e c t r o p h o r e s i s ( 1 8 ) , the p r e s e n t enzyme must t h e r e f o r e be a l m o s t 100% p u r e . The r e s u l t s o f s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f 11S AchE t a k e n b e f o r e and a f t e r 1 month s t o r a g e a t 4°C i n 4 M N a C l b u f f e r a r e shown i n F i g u r e I V - 1 . T h i s f i g u r e shows t h a t t h e fo r m o f t h e enzyme has n o t ch a n g e d due t o a u t o l y s i s d u r i n g s t o r a g e , w h i l e an a s s a y showed t h a t the e n z y m i c a c t i v i t y had n o t d e c r e a s e d s i g n i f i c a n t l y . T h e r e f o r e , l i s AchE seems t o be s t a b l e u nder t h e s e s t o r a g e c o n d i t i o n s . To t e s t f o r a g g r e g a t i o n a t t h e enzyme c o n c e n t r a t i o n u s e d i n t h e NMR e x p e r i m e n t s , a c o n c e n t r a t e d NMR sample was s u b -j e c t e d t o s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . As shown i n F i g u r e I V - 2 , t h e r e i s a s i n g l e s y m m e t r i c peak c o r r e s p o n d i n g t o the 11S - 55 -F i g u r e I V - I : S u c r o s e g r a d i e n t p r o f i l e o f | | S A c h E b e f o r e ( a ) a n d a n d a f t e r ( b ) t h e s t o r a g e i n 4M N a C l b a f f e r a t 4 * C . F i g u r e I V -2: S u c r o s e g r a d i e n t p r o f i l e o f c o n c e n t r a t e d I I S A c h E w h i c h was k e p t i n t h e NMR e x p e r i m e n t a l c o n d i t i o n s . - 5 6 -form. I f the enzyme were aggregated one would e x p e c t t o see a peak skewed towards the l e f t due t o the presence o f enzyme forms w i t h h i g h e r s e d i m e n t a t i o n c o n s t a n t s . T h e r e f o r e , i t can be c o n c l u d e d t h a t the enzyme does not aggregate under the con-d i t i o n s o f the NMR e x p e r i m e n t . F i n a l l y , t o ensure t h a t the enzyme remained a c t i v e d u r i n g the e x p e r i m e n t i t was assayed f o r a c t i v i t y a f t e r s t o r a g e f o r a day a t 17°C and 12 h r s a t 30°C under the c o n d i t i o n o f the NMR e x p e r i m e n t . A l s o , the enzyme appeared t o be s t a b l e i n D 20 b u f f e r a t 4°C f o r up t o 1 week. B. INHIBITORS WITH UNMODIFIED ENZYME The *H NMR s p e c t r a o f a t r o p i n e s u l f a t e (see F i g u r e I V - 3 ) , trimethylammonium c h l o r i d e (TMA) and phenyltrimethylammonium c h l o r i d e (PTA) i n 0.1 M NaCl D 20 b u f f e r (pH 7.0) i n the p r e -sence or absence o f AchE, were examined t o i n v e s t i g a t e the i n t e r a c t i o n o f t h e s e i n h i b i t o r s w i t h the enzyme. A l l these i n h i b i t o r s c o n t a i n m e t h y l g r o u p s , which r e s o n a t e a t 2.68 ppm f o r a t r o p i n e , a t 2.94 ppm f o r TMA, and a t 3.66 ppm f o r PTA d o w n f i e l d from TSP peak r e s p e c t i v e l y . T h e i r p o s i t i o n s d i d not change by more than 0.5 Hz upon a d d i t i o n o f AchE. A l l the s p e c t r a used t o measure l i n e w i d t h s had a s p e c r a l w i d t h o f 100 Hz or 110 Hz depending on the l o c a t i o n of o t h e r peaks f o l d e d i n t o the d i s p l a y e d s p e c t r a l f r e q u e n c y range. The l i n e w i d t h of each CH^ peak was measured a t h a l f maximal h e i g h t from a l i n e - 57 -c i s - 2 , 6 - d i m e t h y l s p i r o -( p i p e r i d i n e - 1 , 1 - p y r r o l i d i u m ) b r o m i d e F i g u r e IV-3: S t r u c t u r e o f a c e t y l c h o l i n e s t e r a s e i n h i b i t o r s u s e d i n NMR e x p e r i m e n t s ' . - 58 -drawn between the f l a t b a s e l i n e s on e i t h e r s i d e of the peak. An a c c u r a c y o f ~ ± 0.05 Hz can be a c h i e v e d by measuring peak w i d t h s w i t h a r u l e r . T h i s t e c h n i q u e o f drawing b a s e l i n e s i s the g r e a t e s t s o u r c e o f l i n e w i d t h e r r o r s , s i n c e the accumula-t i o n o f a l a r g e number of s i g n a l s g i v e s an uneven b a s e l i n e . The most extreme p o s s i b l e v a l u e s o b t a i n e d f o r l i n e w i d t h s o f i n h i b i t o r peaks, depending on how the b a s e l i n e s are drawn, are r e p o r t e d as e r r o r s . The NMR s p e c t r a o f the m e t h y l group i n a t r o p i n e i n the presence and absence of the enzyme are shown i n F i g u r e IV-4» t o g e t h e r w i t h the s p e c t r a of TSP taken b e f o r e and a f t e r accumu-l a t i o n o f the i n h i b i t o r s i g n a l s . Observed changes i n l i n e -w i d t h f o r a t r o p i n e , PTA and TMA a l o n g w i t h the enzyme and i n -h i b i t o r c o n c e n t r a t i o n s are t a b u l a t e d i n Table I V - 1 . AAy i s the symbol used f o r the b r o a d e n i n g of the m e t h y l peak due t o the i n t e r a c t i o n o f i n h i b i t o r w i t h enzyme and i s e x p r e s s e d i n Hz. AAy i s c a l c u l a t e d by t a k i n g the d i f f e r e n c e i n l i n e w i d t h s between a m e t h y l peak and a TSP peak i n the absence of the enzyme and s u b t r a c t i n g i t from the d i f f e r e n c e i n the presence of the enzyme. Here the v a r i a t i o n i n the l i n e w i d t h of the TSP peak i n d i c a t e s the change i n the homogeneity of the magnetic f i e l d d u r i n g measurement. I t was not n e c e s s a r y t o take the b a s e l i n e adjustment i n t o c o n s i d e r a t i o n f o r TSP peak, as i t had an i d e a l f l a t b a s e l i n e . The c o n c e n t r a t i o n o f a c e t y l c h o l i n e s -t e r a s e i s r e p o r t e d as the c o n c e n t r a t i o n o f a c t i v e s i t e s , t a k i n g - 5 9 -F i g u r e IV-4: The ' H NMR s p e c t r a o f a t r o p i n e s u l f a t e w i t h u n m o d i f i e d A c h E . The m e t h y l p e a k s o f a t r o p i n e w i t h ( B ) a n d w i t h o u t (A) a r e shown t o g e t h e r w i t h t h e r e f e r e n c e TSP p e a k s on t h e r i g h t h a n d s i d e , w h i c h w e r e t a k e n b e f o r e a n d a f t e r t h e a c c u m u l a t i o n o f a t r o p i n e s p e c t r a . The o r i g i n a l 100Hz w i d e s p e c t r a h a v e b e e n r e d u c e d by 0 . 7 7 t i m e s f o r t h e a t r o p i n e r e s o n a n c e and by 0 . 2 0 t i m e s f o r t h e TSP r e s o n a n c e . T a b l e IV-1 a t r o p i n e T r i m e t h y l a m m o n i u m c h l o r i d e ( T M A ) P h e n y l t r i m e t h y l -a m m o n i u m c h l o r i d e ( P T A ) C o n c e n t r a t i o n i n h i b i t o r a c e t y i c h o - T e m p e r a t u r e l i n e s t e r a s e ; ( m M ) ( y M ) C ° 0.3 ~ 30 0.3 15 30 0.5 — 30 0.5 14.6 30 0.8 0.8 15 19 19 O b s e r v e d l i n e w i d t h ( H z ) i n h i b i t o r T S P B r o a d e n i n g d u e t o b i n d i n g A A y ( H z ) 1.0±0.1 1.4±0.1 0.5 0.8 0.1+0.14 0.6 0.8±0.1 0.5±0.1 0.7±0.1 0.0±0.2 0.6 0.9±0.1 0.6 0.8±0.1 0.1±0.14 - 61 -80,000 as the m o l e c u l a r weight f o r an a c t i v e s u b u n i t . A l t h o u g h a l l the enzyme used i n the experiment t a k e s the form of l i s , which c o n s i s t s o f f o u r a c t i v e s u b u n i t s , each a c t i v e s u b u n i t i s c o n s i d e r e d as independent s i n c e each u n i t i s known t o be i d e n -t i c a l i n c a t a l y t i c a c t i v i t y and t h e r e i s no c o o p e r a t i v e e f f e c t among s u b u n i t s ( 5 4 ) . I t i s c l e a r from T a b l e IV-1 t h a t t h e r e i s no s i g n i f i c a n t l i n e b r o a d e n i n g (AAy) observed f o r any of the t h r e e . i n h i b i t o r s . The s m a l l l i n e b r o a d e n i n g s (0.1 Hz) observed f o r a t r o p i n e and PTA are s m a l l e r than e x p e r i m e n t a l u n c e r t a i n t y , so i t i s not p o s s i b l e t o observe any l i n e b r o a d e n i n g a t t h i s c o n c e n t r a t i o n or a t h i g h e r c o n c e n t r a t i o n s . These r e s u l t s are unexpected s i n c e Kato (71-74) obse r v e d a c o n s i d e r a b l e l i n e b roadening a t h i g h e r c o n c e n t r a t i o n s o f a t r o p i n e where a s m a l l e r l i n e broaden-i n g i s e x p e c t e d a c c o r d i n g t o e q u a t i o n 4-C i n Table I I - l , where Tjjr- i s dependent on f B which becomes s m a l l e r a t a h i g h e r i n h i b i t o r c o n c e n t r a t i o n . One e x p l a n a t i o n of the p r e s e n t absence of b r o a d -e n i n g i s t h a t the enzyme i s d e n a t u r e d or aggregated and cannot b i n d i n h i b i t o r , g i v i n g no b r o a d e n i n g . However i t was proved i n S e c t i o n A t h a t the enzyme used i n these e x p e r i m e n t s was u n i -form and a c t i v e t h r o u g h o u t , so t h i s cannot be the cause. There are two o t h e r p o s s i b l e c a s e s where one would not observe any l i n e b r o a d e n i n g , even when a c t i v e enzyme i s p r e s e n t . The f i r s t case i s when the system i s i n the slow exchange 1 2 r e g i o n and ^ — 2 > > P B A ' w n e r e the observed l i n e w i d t h i s 2B - 62 -d e s c r i b e d a c c o r d i n g t o the e q u a t i o n g i v e n i n Table I I - l and as f o l l o w s : 1 = J L + f P T 2 T 2 A B BA I f the b i n d i n g c o n s t a n t (K^) f o r the i n h i b i t o r , the i n i t i a l c o n c e n t r a t i o n o f the i n h i b i t o r ( I Q ) and the enzyme c o n c e n t r a -t i o n ( E l are known, the f r a c t i o n of bound i n h i b i t o r (f„) can o a be c a l c u l a t e d by s o l v i n g the f o l l o w i n g q u a d r a t i c e q u a t i o n f o r x x KD where x i s the c o n c e n t r a t i o n of the bound i n h i b i t o r and f D = hi ~ . The c a l c u l a t e d f g v a l u e s f o r a t r o p i n e , PTA and TMA are ° 1 1 a l l 1 - 2 % . When — >> P_ K i s assumed and the v a l u e f o r T 2 B B A T 2 B i s known, the p o s s i b l e range f o r P R A can be e s t i m a t e d . The BA maximum p o s s i b l e v a l u e o f =1— f o r a me t h y l group a t t a c h e d t o a X2B s p h e r i c a l m o l e c u l e i s e v a l u a t e d from F i g u r e I I - l f o r a known r o t a t i o n a l c o r r e l a t i o n time T . For the 11S form o f AchE used i n the p r e s e n t s t u d y , the m o l e c u l a r weight i s 320,000 and the - 3 p a r t i a l s p e c i f i c volume (v) i s 0.72 cm /g ( 2 9 ) . S u b s t i t u t i n g these v a l u e s i n t o e q u a t i o n 6 i n the t h e o r e t i c a l s e c t i o n t o g e t h e r w i t h a v i s c o s i t y (n) o f 0.01 f o r AchE s o l u t i o n , T = 1 x I O - 7 s e c , and ~ - = 400 Hz from F i g u r e I I - l . The l i n e b r o a d e n i n g (AAy) clue t o i n t e r a c t i o n of i n h i b i t o r w i t h enzyme i s the d i f -f e r e n c e between —^— and — , which i s f„P D.. Hence the maximum TT 1 2 ^ 2A exp e c t e d l i n e b r o a d e n i n g i s e s t i m a t e d from the r e l a t i o n s h i p » P B A as f o l l o w s - 6 3 -7 T T 2 B * L B " TT B B A 1 < 4 0 0 H z a n d f „ = 0 . 0 2 T T T 2 B B . * . A A y << 8 H z a n d p e r h a p s A A y < 0 . 1 H z f o r v e r y s l o w e x c h a n g e . H e n c e , n o l i n e b r o a d e n i n g w i l l b e v i s i b l e i f t h e e x c h a n g e r a t e i s t h i s s l o w , s i n c e t h e m a x i m u m r e s o l u t i o n o f t h e N M R s p e c t r a i s 0 . 1 H z . T h e s e c o n d c a s e w h e n n o b r o a d e n i n g i s s e e n i s w h e n t h e s y s t e m i s i n t h e v e r y f a s t e x c h a n g e r e g i o n . H e n c e t h e e x p e c t e d l i n e b r o a d e n i n g i s e x p r e s s e d , a c c o r d i n g t o T a b l e I I - l , a s A A = _ L _ 1 = f 1 " TTT 2 ~ T T T 2 A - R B T T T 2 B a n d t h e m a x i m u m p o s s i b l e v a l u e f p , - 7 j r — c a n t a k e i s 8 H z w h e n B l T i 2 B — = — i s 4 0 0 H z . I f t h e i n h i b i t o r i s l o o s e l y b o u n d t o t h e e n -t t T 2 B z y m e o r t h e m e t h y l g r o u p o n t h e i n h i b i t o r s p i n s f r e e l y , — = — w i l l b e s m a l l e r . W h e n — = — i s l e s s t h a n 5 H z , t h e e x p e c t e d 7 T T 2 B l i n e b r o a d e n i n g w i l l b e l e s s t h a n 0 . 1 H z . T h e r e f o r e i t i s p o s -s i b l e n o t t o o b s e r v e a n y l i n e b r o a d e n i n g i f t h e s y s t e m i s i n v e r y f a s t r e g i o n i f t h e l a b e l e d s i t e i s v e r y f l e x i b l e . I t m i g h t b e w o r t h w h i l e n o t i c i n g t h a t t h e o n l y c o m p o u n d , t h o u g h i t h a s a c o u p l e d m e t h y l p e a k , w h i c h g a v e s i g n i f i c a n t b r o a d e n i n g a t a n i n h i b i t o r c o n c e n t r a t i o n o f 0 . 5 mM w a s c i s - 2 , 6 - d i m e t h y l s p i r o -( p i p e r i d i n e - 1 , 1 1 - p y r r o l i d i u m ) b r o m i d e ( s t r u c t u r e s h o w n i n F i g u r e I V - 3 ) . E x a c t v a l u e s o f h e n c e TJT—, a r e n o t o b t a i n a b l e h o w -l2 2 B e v e r d u e t o t h e m u l t i p l e t s t r u c t u r e o f t h e p e a k s . - 6 4 -C . E S E R I N E W I T H C A R B A M Y L A T E D E N Z Y M E T h e b i n d i n g o f e s e r i n e ( s e e F i g u r e I V - 3 ) t o c a r b a m y l a t e d e n z y m e w a s s t u d i e d b y m e a s u r i n g t h e c h a n g e i n l i n e w i d t h o f C - m e t h y l g r o u p , w h i c h r e s o n a t e s a t 1 . 5 1 p p m d o w n f i e l d f r o m T S P p e a k . T h e s e q u e n t i a l a d d i t i o n o f e s e r i n e s u l f a t e s t o c k s o l u t i o n t o t h e e n z y m e s o l u t i o n , i n t h e s a m e b u f f e r a s i n S e c t i o n B , p r o d u c e d t h e t y p i c a l s p e c t r a s h o w n i n F i g u r e I V - 5 . T h e C - m e t h y l g r o u p r e s o n a n c e s o f e s e r i n e a t 3 0 ° C ( p H 7 . 0 ) a r e m a r k e d l y b r o a d e n e d i n t h e p r e s e n c e o f t h e e n z y m e . H o w e v e r , w i t h i n e x p e r i m e n t a l e r r o r ( e s t i m a t e d t o b e l e s s t h a n 1 % ) , n o s i g n i f i c a n t c h a n g e s i n t h e r e s o n a n c e f r e q u e n c y o f t h e s e l i n e s c o u l d b e o b s e r v e d i n t h e p r e s e n c e o f A c h E . T h e p o s s i b i l i t y t h a t t h e s y s t e m i s i n s l o w e x c h a n g e r e g i o n c a n b e e x c l u d e d s i n c e n o l i n e b r o a d e n i n g i s e x p e c t e d i n t h i s r e g i o n a s d e s c r i b e d i n S e c t i o n B . I f t h e e x c h a n g e r a t e i s f a s t o r v e r y f a s t , t h e p e a k p o s i t i o n i s e x p e c t e d t o m o v e t o t h e f r e q u e n c y w h i c h i s t h e w e i g h t e d a v e r a g e o f t h e p o s i t i o n f o r t h e b o u n d a n d u n b o u n d s p e c i e s ( 8 2 ) . H o w e v e r , b e c a u s e a s h i f t o f l e s s t h a n 1 H z w a s o b s e r v e d , t h e d i f f e r e n c e i n c h e m i c a l s h i f t b e t w e e n t h e b o u n d a n d f r e e m e t h y l g r o u p s m u s t b e l e s s t h a n 5 0 H z w h e n g r e a t e r t h a n 2 % o f t h e i n h i b i t o r m o l e c u l e s a r e b o u n d . T h i s f i n d i n g i s n o t s u r p r i s i n g s i n c e m o s t i n h i b i t o r s o f o t h e r e n z y m e s d o n o t p r o d u c e c h a n g e s i n c h e m i c a l s h i f t w h e n b o u n d . T h e e x c e p t i o n i s f o r i n h i b i t o r s o f l y s o z y m e w h i c h h a s a d e -s h i e l d i n g p h e n y l g r o u p n e a r t h e b i n d i n g s i t e ( 9 7 ) . I n t h e f a s t - 6 5 -F i g u r e I V - 5 : The 'H NMR s p e c t r a o f e s e r i n e s u l f a t e w i t h c a r b a m y l a t e d A c h E . The C - m e t h y l p e a k s o f e s e r i n e a r e shown i n t h e f i r s t c o l u m n i n o r d e r o f i n c r e a s i n g r a t i o o f e s e r i n e t o A c h E f r o m t o p t o b o t t o m . The s p e c t r a i n t h e b o t t o m row w e r e o b t a i n e d i n t h e a b s e n c e o f A c h E . A l s o shown a r e t h e r e f e r e n c e TSP p e a k s t a k e n b e f o r e ( s e c o n d c o l u m n ) a n d a f t e r ( l a s t c o l u m n ) t h e a c c u m u l a t i o n o f e s e r i n e s p e c t r a . The o r i g i n a l 120Hz w i d e s p e c t r a h a v e b e e n r e d u c e d 0 . 2 6 t i m e s . - 66 -exchange case P n , >> Aio as g i v e n i n Table I I - l . I t f o l l o w s f B A a ) 2 t h a t i n t h i s r e g i o n the ex p e c t e d l i n e b roadening — 5 (Table BA I I - 2 ) i s much l e s s than fgAco. However ALO i s c a l c u l a t e d t o be l e s s than 50 Hz and f_ i s 2%. T h e r e f o r e i t can be s a i d t h a t i f the exchange r a t e i s f a s t the l i n e b r o a d e n i n g would be much s m a l l e r than 1 Hz and would not be o b s e r v a b l e . S i n c e one e x p e c t s t o see no l i n e b r o a d e n i n g when the system i s i n the slow or f a s t exchange r e g i o n s , the l i n e b roadening observed f o r e s e r i n e must be c o n t r o l l e d by the T 2 B r e l a x a t i o n p r o c e s s and the exchange r a t e must be v e r y f a s t . There are two methods t o deduce the v a l u e of =r- f o r the bound i n h i b i t o r when the system i s i n the v e r y f a s t exchange r e g i o n . As mentioned i n the t h e o r y s e c t i o n both methods have d i s a d v a n t a g e s . In o r d e r t o use the e x p r e s s i o n by G e r i g ( E q u a t i o n 7 ) ; E AAy = I ° • A y E I (9) o D the v a l u e o f K_ must be known. Here (7^ - - and — are D T 2 T 2 A TT T T T 2 B r e p l a c e d by AAy a n ( 3 A y E I • A l t h o u g h f o r e s e r i n e b i n d i n g t o - 6 AchE (3.3 x 10 M) (46) i s much s m a l l e r than I i n the p r e s e n t e x p e r i m e n t , t h i s v a l u e i s expected t o be l a r g e r f o r ca r b a m y l a t e d AchE and can n o t s i m p l y be assumed t o be n e g l i g i b l e compared w i t h I when an e x a c t v a l u e of K D f o r c a r b a m y l a t e d AchE i s not known. In the o t h e r method, the v a l u e of 7^ - f o r the bound i n -h i b i t o r may be o b t a i n e d w i t h o u t knowing the v a l u e o f K D, by u s i n g i n s t e a d the e x p r e s s i o n by K a t o ( E q u a t i o n 8) - 67 -J o = E o ^ F - K n ( 1 0 ) o o AAy u However the assumption made t o d e r i v e t h i s e q u a t i o n ; namely 1 >> •—-, may not be r e a s o n a b l e i n the p r e s e n t c a s e . m ' ' m The observed l i n e b r o a d e n i n g s ( A A Y = "Tfr- ~ ~ ^ — ) a r ^ TTT 2 T T T 2 B p l o t t e d a c c o r d i n g t o both e q u a t i o n s as seen i n F i g u r e IV-6 and _ g F i g u r e IV-7. A K D v a l u e of 3.3 x 10 M f o r e s e r i n e b i n d i n g t o AchE i s used t o o b t a i n F i g u r e IV-6. The same methods f o r the c a l c u l a t i o n of AAy a n <3 the e v a l u a t i o n of the e r r o r as d e s c r i b e d i n S e c t i o n B are used h e r e . From the s l o p e s o f b e s t f i t t i n g s t r a i g h t l i n e s o b t a i n e d by w e i g h t e d * l e a s t square f i t i n F i g u r e IV-6 and IV-7, the l i n e w i d t h s o f a C-methyl peak o f e s e r i n e bound t o the c a r b a m y l a t e d AchE are determined t o be 273 + 23 Hz and 248 + 29 Hz r e s p e c t i v e l y . The e r r o r note here i s the s t a n d a r d d e v i a t i o n o f s t r a i g h t l i n e s g o i n g t h rough p l o t t e d p o i n t s . The v a l u e s o f Ay E I o b t a i n e d from two methods agree w i t h i n e x p e r i m e n t a l e r r o r s . The i n t e r c e p t of the s l o p e from F i g u r e IV-6 i s c a l c u l a t e d t o be 0.0 ± 0.1 and so the s t r a i g h t l i n e goes through the o r i g i n as e x p e c t e d . F i g u r e IV-7 g i v e s the c a l c u l a t e d i n t e r c e p t of -115 ± 168 yM, which r e p r e s e n t s the K Q v a l u e of e s e r i n e i n t e r a c t i n g w i t h c a r b a m y l a t e d W e i g h t i n g f a c t o r i s c a l c u l a t e d as f o l l o w s : 10 - l e n g t h of e r r o r bar .max v a l u e - min v a l u e . ( 30 ' - 68 -F i g u r e I V - 6 : A p l o t o f M i V f o r t h e C - m e t h y l p r o t o n r e s o n a n c e s o f e s e r i n e w i t h r e s p e c t t o E Q / ^ - K Q . F i g u r e I V - 7 : A p l o t o f t h e r e c i p r o c a l o f A A V f o r t h e C - m e t h y l r e s o n a n c e s o f e s e r i n e v s . v a r y i n g c o n c e n t r a t i o n s o f e s e r i n e s u l f a t e . F i g u r e s I V - 6 a n d I V - 7 w e r e p l o t t e d a c c o r d i n g t o e q u a t i o n s 9 a n d 10 r e s p e c t i v e l y , AAV b e i n g t h e l i n e b r o a d e n i n g d e d u c e d f r o m t h e o b s e r v e d l i n e w i d t h s as d e s c r i b e d i n t e x t . A l l s p e c t r a f o r a b o v e f i g u r e s w e r e r e c o r d e d a t 100MHz f i e l d s t r e n g t h , 30 C , pH 7 . 0 . - 6 9 -AchE. However, as a n t i c i p a t e d from Chapter I I I , the i n t e r c e p t c a l c u l a t e d ( 1 1 5 ± 1 6 8 yM) i s not v e r y u s e f u l s i n c e the K D v a l u e i s e x p e c t e d t o be s m a l l e r than 1 0 0 yM. From the v a l u e o b t a i n e d f o r A y E I ' i t c a n be s a i d t h a t the assumption made t o o b t a i n F i g u r e I V - 7 , namely —=r— >> - ~ — , i s r e a s o n a b l e . A l s o 7 T T 2 B T T T 2 A K D f o r c a r b a m y l a t e d AchE would not have been g r e a t l y d i f f e r e n t from one f o r n a t i v e AchE, as the s t r a i g h t l i n e goes through the o r i g i n and the agreement of A y E j v a l u e s o b t a i n e d by two methods i s good. When Kato c a l c u l a t e d the l i n e w i d t h of bound e s e r i n e , he assumed e s e r i n e was a r e v e r s i b l e i n h i b i t o r and was i n t e r a c t i n g w i t h n a t u r a l enzyme ( 7 1 , 7 2 , 7 3 ) . However, i t has been r e p o r t e d by o t h e r s t h a t e s e r i n e i n h i b i t s AchE i r r e v e r s i b l y by c a r b a m y l a t -i n g the c a t a l y t i c s e r i n e r e s i d u e ( 4 6 , 1 0 8 , Chapter I I ) and e s e r -ine i s now w i d e l y a c c e p t e d as an i r r e v e r s i b l e i n h i b i t o r of AchE w i t h a i n h i b i t i o n r a t e c o n s t a n t k^ v a l u e of 3.3 x 1 0 M ^ min f o r the r e a c t i o n k . EH + PX EP +• XH In the above r e a c t i o n EH, PX, EP and XH r e p r e s e n t AchE, e s e r -i n e , c a r b a m y l a t e d AchE and e s e r o l i n e (a h y d r o l y s i s p r o d u c t of e s e r i n e ) r e s p e c t i v e l y . The time r e q u i r e d t o c a r b a m y l a t e 9 9 % of the AchE a t the c o n c e n t r a t i o n s of enzyme and e s e r i n e used i n the ex p e r i m e n t i s c a l c u l a t e d t o be 1.6 x 1 0 5 min. I t i s hence j u s t i f i e d t o c o n c l u d e t h a t a l l the changes observed i n - 70 -the NMR l i n e w i d t h o f the m e t h y l group are due t o the i n t e r a c -t i o n o f e s e r i n e w i t h c a r b a m y l a t e d AchE and not w i t h n a t u r a l A c h E . A l t h o u g h the k^ v a l u e f o r e s e r i n e i n h i b i t o r i s w e l l e s t a b l i s h e d , t h e r e are d i s c r e p a n c i e s among the v a l u e s o f the r e c o v e r y r a t e o f i n h i b i t e d AchE (46 and p e r s o n a l communication w i t h Dr. R o u f o g a l i s ) . I f the enzyme r e c o v e r s e f f i c i e n t l y t o produce enough e s e r o l i n e , the m e t h y l peak i n e s e r o l i n e w i l l i n t e r f e r e w i t h the one i n e s e r i n e . In f a c t the s u p e r p o s i t i o n o f two peaks might appear t o produce b r o a d e n i n g of the peak when the resonance p o s i t i o n s of the two peaks are c l o s e t o -g e t h e r . T h i s was indeed the case when the b i n d i n g o f a c e t y l -c h o l i n e t o AchE was s t u d i e d through the change i n NMR l i n e w i d t h of the m e t h y l group of A c e t y l c h o l i n e (99,100). In o r d e r t o t e s t i f the c h e m i c a l s h i f t s o f the m e t h y l groups of e s e r o l i n e and e s e r i n e are near enough t o g e t h e r t o produce o v e r l a p p i n g peaks, e s e r o l i n e was p r e p a r e d as i n Chapter I I I and i t s NMR spectrum was o b t a i n e d i n the same b u f f e r s o l u t i o n as was used i n the b i n d i n g e x p e r i m e n t s . The C-methyl group of e s e r o l i n e was found t o r e s o n a t e a t 1.46 ppm d o w n f i e l d from TSP, a f u l l 5 Hz away from the C-methyl resonance o f e s e r i n e . T h e r e f o r e , i t i s im-p o s s i b l e f o r the two m e t h y l resonances t o be superimposed and the o b s e r v e d l i n e b r o a d e n i n g i s indeed due t o the i n t e r a c t i o n between e s e r i n e and c a r b a m y l a t e d AchE. - 71 -P. INHIBITORS WITH METHANESULFONYLATED AchE The r e s u l t s o b t a i n e d i n S e c t i o n C i n d i c a t e t h a t the modi-f i c a t i o n o f AchE a t the c a t a l y t i c s i t e a l l o w s one t o observe the b r o a d e n i n g of NMR peaks e i t h e r by a l t e r i n g the exchange r a t e or the bound l i n e w i d t h o f the i n h i b i t o r . Some r e v e r s i b l e i n h i b i t o r s are known t o b i n d more p o o r l y t o the m o d i f i e d AchE than t o the n a t u r a l AchE (26,46 ,76,92) and c o n s e q u e n t l y the o f f r a t e s from m o d i f i e d AchE f o r t h e s e i n h i b i t o r s are e x p e c t e d t o be f a s t e r . T h i s h y p o t h e s i s was t e s t e d by l e t t i n g the same i n -h i b i t o r s as used f o r n a t u r a l enzyme ( a t r o p i n e , TMA, PTA) i n t e r -a c t w i t h m o d i f i e d enzyme and comparing the l i n e w i d t h o f t h e i r m e t h y l peaks f o r the two enzyme forms. The s u l f o n y l a t e d AchE, f r e e o f m e t h a n e s u l f o n y l f l u o r i d e , i n D 20 b u f f e r i s p r e p a r e d as d e s c r i b e d i n Chapter I I I . As can be seen from m e t h y l peaks of TMA i n F i g u r e IV-8 t o g e t h e r w i t h TSP peak, c o n t r a r y t o expec-t a t i o n no s i g n i f i c a n t l i n e b r o a d e n i n g was observed (AAy = 0.2 ± 0.1 Mz) when the c o n c e n t r a t i o n of TMA and m o d i f i e d AchE are 0.5 mM and 18.6 uM r e s p e c t i v e l y . However the C-methyl peaks of a t r o p i n e and PTA do indeed broaden when these i n h i b i t o r s i n t e r a c t w i t h m o d i f i e d AchE. T h i s can be seen i n F i g u r e s IV-9 and IV-10, where t y p i c a l s p e c t r a 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 o f i n h i b i t o r s are shown. The o b s e r v e d l i n e b r o a d e n i n g s (AAy) are measured i n the same manner as d e s c r i b e d i n S e c t i o n B. I t was assumed t h a t the system was i n the v e r y f a s t exchange r e g i o n f o r the same reason - 72 -F i g u r e I Y - 8 : The 'H NMR s p e c t r a o f t r i m e t h y l a m i n e h y d r o c h l o r i d e w i t h m o d i f i e d A c h E . The m e t h y l p e a k s o f t r i m e t h y l a m m o n i u m i n t h e a b s e n c e ( A ) a n d ( B ) o f s u l f o n y l a t e d A c h E a r e shown t o g e t h e r w i t h t h e r e f e r e n c e p e a k s on t h e r i g h t h a n d s i d e . The o r i g i n a l 100 Hz w i d e s p e c t r a h a v e b e e n r e d u c e d 0 . 4 5 t i m e s . - 73 -F i g u r e I V - 9 : The H NMR s p e c t r a o f a t r o p i n e s u l f a t e w i t h m o d i f i e d A c h E . The m e t h y l p e a k s o f a t r o p i n e a t t h e l e f t h a n d s i d e a r e shown i n o r d e r o f i n c r e a s i n g r a t i o o f t h e i n h i b i t o r t o t h e s u l f o n y l a t e d A c h E f r o m t o p t o b o t t o m . C o r r e s p o n d i n g r e f e r e n c e TSP p e a k s a r e shown t o t h e r i g h t . A l l s p e c t r a shown a b o v e a r e t h e o r i g i n a l 2 0 0 H z w i d e s p e c t r a r e d u c e d 0 . 2 6 t i m e s . - 74 -F i g u r e I V - 1 0 : The 'H NMR s p e c t r a o f p h e n y l t r i m e t h y l ammonium c h l o r i d e w i t h m o d i f i e d A c h E . The m e t h y l p e a k s o f p h e n y l t r i m e t h y l -ammonium ( f a r l e f t ) a r e shown t o g e t h e r w i t h t h e r e f e r e n c e TSP p e a k s ( t o t h e r i g h t ) . The s p e c t r a a r e a r r a n g e d f r o m t o p t o b o t t o m i n o r d e r o f i n c r e a s i n g i n h i b i t o r t o s u l f o n y l a t e d A c h E r a t i o , a n d t h e b o t t o m s p e c t r a w e r e o b t a i n e d i n t h e a b s e n c e o f e n z y m e . A l l s p e c t r a shown a b o v e a r e t h e o r i g i n a l 60Hz w i d e s p e c t r a r e d u c e d .31 t i m e s . - 75 -as g i v e n i n S e c t i o n C i n o r d e r t o p l o t the observed l i n e b r o a d e n i n g a c c o r d i n g t o e q u a t i o n s (9) and (10) . The p l o t t e d graphs are shown i n F i g u r e s IV-11, IV-12 and IV-13 u s i n g a v a l u e K D = 5.3 x 1 0 ~ 5 M f o r PTA b i n d i n g t o n a t u r a l AchE ( 4 6 ) . For a t r o p i n e o n l y e q u a t i o n (10) i s used f o r p l o t t i n g as the K D v a l u e f o r a t r o p i n e has not been measured. The bound l i n e -w i d t h s ( A Y e i ) a re o b t a i n e d i n the same manner d e s c r i b e d i n S e c t i o n C. They are found t o be 14 ± 1 Hz and 16 + 2 Hz f o r the PTA m e t h y l peak and 97 ± 11 Hz f o r the a t r o p i n e m e t h y l peak. The agreement o f the two v a l u e s of Aygj o b t a i n e d f o r PTA i s e s p e c i a l l y good c o n s i d e r i n g t h a t two c o n d i t i o n s r e q u i r e d t o use e q u a t i o n s (9) and ( 1 0 ) , namely I Q >>KD ( f o r n a t u r a l en-zyme) and A y E I >> Ayj» a r e n o t s a t i s f i e d . However these c o n d i -t i o n s may be the reason why the p l o t t e d graph i n F i g u r e IV-13 l o o k s more l i k e a h y p e r b o l a than a s t r a i g h t l i n e and g i v e s a p o s i t i v e i n t e r c e p t , which i m p l i e s a n e g a t i v e K D» A l s o the s t r a i g h t l i n e i n F i g u r e IV-12 does not go through the o r i g i n . In S e c t i o n s B and C a l l *H s p e c t r a were r e p o r t e d by u s i n g a V a r i a n XL100 NMR s p e c t r o m e t e r . In t h i s s e c t i o n most of the s p e c t r a were o b t a i n e d by a NIC-270 FT NMR s p e c t r o m e t e r e x c e p t the ones f o r a t r o p i n e . The l i n e w i d t h s of a t r o p i n e m e t h y l peaks were observed a t 20°C and 30°C u s i n g a V a r i a n XL100 and a t 20°C u s i n g a 270 MHz s p e c t r o m e t e r . When the temperature i s d e c r e a s e d from 30°C t o 20°C, a 0.2 Hz broad e n i n g of the l i n e i s o b s e r v e d . The r e v e r s e i s expected i n the slow exchange r e g i o n - 76 -E 0 / A A V ( « M / H Z ) F i g u r e I V - 1 1 : A p l o t o f t h e r e c i p r o c a l o f LhV f o r t h e m e t h y l g r o u p r e s o n a n c e s w i t h r e s p e c t t o v a r y i n g c o n c e n t r a t i o n s o ^ a t r o p i n e s u l f a t e ( I Q ) . A c o r r e s p o n d s t o t h e s p e c t r u m r e c o r d e d a t 100MHz f i e l d s t r e n g t h , 2 0 ° C . The r e m a i n i n g p o i n t s w e r e o b t a i n e d f r o m s p e c t r a o b t a i n e d a t 2 7 0 M H z , 2 0 ° C . F i g u r e I v - 1 2 : A p l o t o f LLV f o r t h e m e t h y l p r o t o n r e s o n a n c e s o f p h e n y l -t r i m e t h y l a m m o n i u r n w i t h r e s p e c t t o E Q / ^ Q - K Q . F i g u r e I V - 1 3 : A p l o t o f r e c e p r o c a l o f AA.V f o r t h e m e t h y l g r o u p r e s o n a n c e s o f p h e n y l t r i m e t h y l a m m o n i u r n v s . c o n c e n t r a t i o n . F i g u r e s I V - 1 2 a n d I V - 1 3 w e r e p l o t t e d a c c o r d i n g t o e q u a t i o n s 9 a n d 10 r e s p e c t i v e l y . A l l s p e c t r a f o r a b o v e f i g u r e s w e r e r e c o d e d a t 270MHz f i e l d s t r e n g t h , 2 0 6 C , pH 7 . 0 . - 78 -s i n c e the d e c r e a s e i n temperature w i l l lower the o f f r a t e ( P B A ) i n the e q u a t i o n i n T a b l e I I - l and g i v e s m a l l e r A l s o the l i n e b r o a d e n i n g o b s e r v e d a t 20°C w i t h XL100 ( i n d i c a t e d w i t h L j f i t w e l l as the s t r a i g h t l i n e of F i g u r e IV-11 ob-t a i n e d by the 270 MHz s p e c t r o m e t e r . I f the exchange r a t e i s i n the f a s t r e g i o n the l i n e b r o a d e n i n g due t o the i n t e r a c t i o n i s g i v e n a c c o r d i n g t o the e q u a t i o n i n Table I I - l A A 1/1 __1 \ _ -F (Acof ^ i2 2A B T r ± B A The change i n magnetic f i e l d s t r e n g t h from 270 MHz t o 100 Hz w i l l d e c r e a s e Aco and hence AAy by 1/7.3 t i m e s . Hence i f the system was i n the f a s t exchange r e g i o n , the v a l u e of E o/AAy, o b t a i n e d w i t h a XL100, would have been 7.3 t i m e s g r e a t e r than ones o b t a i n e d w i t h 270 MHz s p e c t r o m e t e r and would be l o c a t e d f u r t h e r t o the r i g h t from s t r a i g h t l i n e . These two o b s e r v a -t i o n s c o n f i r m the s p e c u l a t i o n made e a r l i e r t h a t the exchange r a t e can not be e i t h e r i n the slow or the f a s t exchange r e g i o n . - 79 -CHAPTER V DISCUSSION A. INHIBITORS WITH UNMODIFIED AchE Expe r i m e n t s s i m i l a r t o tho s e d e s c r i b e d i n Chapter IV were c a r r i e d out by Kato , and showed c o n s i d e r a b l e broadening f o r both the a t r o p i n e and e s e r i n e peaks i n the presence of AchE, even a t i n h i b i t o r c o n c e n t r a t i o n s 100 times h i g h e r than i n the p r e s e n t work (71,72,74). Kato's v a l u e s o b t a i n e d f o r the bound l i n e -w i d t h of both the a t r o p i n e N-methyl and e s e r i n e C-methyl peaks are i n c o n s i s t e n t , b e i n g 21,000, 5,600 and 984 Hz f o r a t r o p i n e , and 13,000 and 488 Hz f o r e s e r i n e . They are a l l too l a r g e , as l i n e w i d t h s f o r s m a l l m o l e c u l e s t i g h t l y bound t o 11S AchE s h o u l d not exceed 400 Hz, as e s t i m a t e d i n Chapter IV. To c l a r i f y t h i s , the experiment was r e p e a t e d u s i n g the same i n h i b i t o r s as Kato. S m a l l e r bound l i n e w i d t h and hence s m a l l e r l i n e b r o a d e n i n g were e x p e c t e d . Our r e s u l t s i n d i c a t e d no t r a c e of l i n e b roadening w i t h a l l i n h i b i t o r s e x c e p t c i s - 2 , 6 - D i m e t h y l s p i r o - ( p i p e r i d i n e -1 , 1 ' - p y r r o l i d i u m ) b r o m i d e . The exchange r a t e can be i n e i t h e r the slow or the f a s t exchange r e g i o n but the c h e m i c a l s h i f t d i f f e r e n c e between bound and f r e e i n h i b i t o r s i s s m a l l . Even w i t h the system i n the v e r y f a s t exchange r e g i o n , l i n e b r o a d -eni n g w i l l not be v i s i b l e i f the bound i n h i b i t o r i s f l e x i b l e . Other f a c t o r s such as d e n a t u r a t i o n and a g g r e g a t i o n which might cause non-broadening by i n t e r f e r i n g w i t h i n h i b i t o r s b i n d i n g t o - 80 -the enzyme, are r i g o r o u s l y e x c l u d e d i n the p r e v i o u s c h a p t e r , as i s the p o s s i b l e i n f l u e n c e o f t r y p s i n and soybean t r y p s i n i n h i b i t o r p r e s e n t i n the enzyme e x t r a c t . These p r o t e i n s were found not t o a f f e c t the c a t a l y t i c a c t i v i t y of a c e t y l c h o l i n e s t e r -ase, s u g g e s t i n g t h a t n e i t h e r i n t e r f e r s w i t h the i n h i b i t o r -enzyme b i n d i n g . The reason f o r the observed non-broadening o f the NMR peaks i n the presence of AchE can hence be b e s t ex-p l a i n e d by any o f the f o l l o w i n g c a s e s . 1) The exchange r a t e i s s l o w , w i t h the c o n d i t i o n >> P t , , 2B a A b e i n g met. 2) The exchange r a t e i s f a s t ( a l t h o u g h u n l i k e l y ) , w i t h a s m a l l c h e m i c a l s h i f t d i f f e r e n c e between f r e e and bound s p e c i e s (Aco) , r e s u l t i n g i n no o b s e r v a b l e l i n e b r o a d e n i n g . 3) The exchange r a t e i s v e r y f a s t , where l i n e b r o a d e n i n g may not be o b s e r v a b l e a t the c o n c e n t r a t i o n o f i n h i b i t o r s and enzyme used, p r o v i d e d t h a t the bound i n h i b i t o r i s f l e x i b l e . A l l c a s e s were d e s c r i b e d i n d e t a i l i n S e c t i o n B and C o f the p r e v i o u s c h a p t e r . The o b v i o u s d i s c r e p a n c y between the bound l i n e w i d t h s found by Kato and the ones measured i n t h i s work, can be l a r g e l y e x p l a i n e d by the d i f f e r e n c e i n p r e p a r a t i o n of the enzyme ex-t r a c t (and hence the c o n d i t i o n of the enzyme). He r e p o r t e d an enzyme w i t h a s p e c i f i c a c t i v i t y of 3.3 mmoles of a c e t y l c h o -l i n e h y d r o l y z e d per min per mg of p r o t e i n , c o r r e s p o n d i n g t o a p u r i t y o f a p p r o x i m a t e l y 30%, assuming t h a t s q u i d head AchE i s - 81 -s i m i l a r t o e e l enzyme. With such a low p u r i t y enzyme, the pos-s i b i l i t y of n o n - s p e c i f i c b i n d i n g has t o be taken i n t o c o n s i d e r a -t i o n , which c o u l d a l s o c o n t r i b u t e t o the b r o a d e n i n g o f the l i n e w i d t h s . S e c o n d l y , s q u i d head AchE i s l e s s w i d e l y s t u d i e d than e e l enzyme, and i t i s not known w i t h c e r t a i n t y whether the a g g r e g a t i v e p r o p e r t i e s are s i m i l a r . I t i s p r o b a b l e t h a t the enzyme used by Kato was aggregated s i n c e the enzyme e x t r a c t con-t a i n e d no s a l t . The e f f e c t of the s i z e o f the enzyme on the bound l i n e w i d t h can be r e a d i l y seen from F i g u r e I I - l , and the T c v a l u e c a l c u l a t e d from the assumed m o l e c u l a r w e i g h t . A t e n -f o l d i n c r e a s e i n m o l e c u l a r weight w i l l thus i n c r e a s e t c and hence the bound l i n e w i d t h ( ) by the same amount. T h e r e f o r e , i t i s r e a s o n a b l e t o suggest t h a t Kato's enormous bound l i n e -w i d t h c o u l d be p a r t l y caused by enzyme a g g r e g a t i o n . S q u i d head and e l e c t r i c e e l AchE c o u l d a l s o p o s s i b l y e x h i b i t d i f f e r e n t b i n d i n g modes, hence a l t e r i n g —-, a l t h o u g h i t i s u n l i k e l y con-s i d e r i n g the s i m i l a r i t y i n i n h i b i t o r s p e c i f i c i t y and c a t a l y t i c a c t i v i t y among the AchE from d i f f e r e n t s o u r c e s . Other f a c t o r s t o o , b e s i d e s the c o n d i t i o n of the enzyme i t s e l f , can l e a d t o a d d i t i o n a l l i n e b r o a d e n i n g . These i n c l u d e magnetic inhomogen-e i t y , i n c r e a s e d v i s c o s i t y , or the presence of i m p u r i t i e s such as paramagnetic i o n s . Even when the above mentioned s o u r c e s of anomalous l i n e b r o a d e n i n g are e l i m i n a t e d , Kato's r e s u l t s are not e s p e c i a l l y s i g n i f i c a n t , due t o the n a t u r e o f the enzyme. The f r e e z e - d r i e d - 82 -AchE used may n o t have the same form as f r e s h enzyme, s i n c e i t i s known t h a t a g e i n g has a marked e f f e c t on the enzyme a c t i v i t y ( 4 6 ) . The enzyme was a l s o n ot w e l l c h a r a c t e r i z e d , and most l i k e l y c o m p r i sed o f s e v e r a l d i f f e r e n t forms o f AchE. Homogen-e i t y o f the enzyme i s a l s o e s s e n t i a l , s i n c e the bound l i n e w i d t h of i n h i b i t o r s depends upon the s i z e of the enzyme. T h e r e f o r e o n l y i f the enzyme i s p r e p a r e d under c o n t r o l l e d , r e p r o d u c i b l e c o n d i t i o n s , c o n s i s t e n t v a l u e s can be e x p e c t e d f o r 7—-. To o v e r -come the s e problems, o n l y the l i s form of AchE was used i n the p r e s e n t work, w i t h the f o l l o w i n g advantages; the 11S form i s s t a b l e , non a g g r e g a t i v e , and a l s o g l o b u l a r , which made i t pos-s i b l e t o e s t i m a t e the maximum bound l i n e w i d t h f o r an i n h i b i t o r u s i n g F i g u r e I I - l . The 11S s p e c i e s i s the most s t u d i e d form o f AchE, w i t h a c a t a l y t i c a c t i v i t y i d e n t i c a l t o the n a t i v e 18S form. The b i n d -i n g of i n h i b i t o r s t o the 11S t e t r a m e r i s s t o i c h i o m e t r i c t o i t s 80,000 M.W. c a t a l y t i c s u b u n i t s , w i t h the same b i n d i n g c o n s t a n t K j . As no c o - o p e r a t i v i t y i s observed among the s u b u n i t mono-mers, t h e y a r e c o n s i d e r e d t o c o n t a i n independent i d e n t i c a l b i n d i n g s i t e s w i t h r e s p e c t t o i n t e r a c t i o n w i t h i n h i b i t o r s . The c l a i m t h a t t h i s enzyme s p e c i e s i s the most s u i t a b l e form t o s t u d y , i s a l s o s u p p o r t e d by i t s w i d e s p r e a d use i n r e c e n t s p e c t r o s c o p i c i n v e s t i g a t i o n s on a c e t y l c h o l i n e s t e r a s e (39,76,77). - 83 -B. INHIBITORS WITH MODIFIED ENZYME The i n t e r a c t i o n of e s e r i n e , a t r o p i n e and PTA (TMA i s an e x c e p t i o n ) w i t h the m o d i f i e d enzyme produces a s i g n i f i c a n t b r o a d e n i n g o f the NMR re s o n a n c e s . S i n c e t h e r e are s e v e r a l p o s s i b l e r e a s o n s ( b e s i d e s the e n z y m e - i n h i b i t o r i n t e r a c t i o n ) t h a t c o u l d cause the l i n e b r o a d e n i n g , the f a c t t h a t t h e r e i s no d e t e c t a b l e l i n e b r o a d e n i n g w i t h n a t u r a l enzyme has t o be c o n s i d e r e d an e x c e l l e n t c o r r o b o r a t i o n of the a p p l i e d experimen-t a l c o n d i t i o n s , s u g g e s t i n g the observed l i n e b roadening w i t h the m o d i f i e d enzyme i s indeed due t o e n z y m e - i n h i b i t o r i n t e r a c t i o n s . The bound l i n e w i d t h s , which were found f o r the i n t e r a c t i o n of the t h r e e i n h i b i t o r s w i t h the s u l f o n y l a t e d enzyme (260 Hz f o r e s e r i n e , 15 Hz f o r PTA, and 98 Hz f o r a t r o p i n e ) are a l s o r e a s o n -a b l e and w i t h i n the p r e v i o u s l y c a l c u l a t e d l i m i t of 400 Hz. As d e s c r i b e d i n the p r e v i o u s s e c t i o n the non-broadening of the i n h i b i t o r peaks r e s u l t s e i t h e r from a f a s t or a slow exchange r a t e , or from a v e r y f a s t exchange w i t h a v e r y f l e x -i b l e b i n d i n g o f the i n h i b i t o r . S u l f o n y l a t i o n o f AchE must t h e r e f o r e have a l t e r e d e i t h e r the exchange r a t e or the f l e x i -b i l i t y of the i n h i b i t o r when bound. L e t us f i r s t c o n s i d e r the p o s s i b i l i t y o f a v a r i a t i o n i n the exchange r a t e . There w i l l be two arguments used i n orde r t o i n v e s t i g a t e i f the obser v e d s p e c t r a l d i f f e r e n c e between systems w i t h n a t u r a l and m o d i f i e d enzyme was due t o the a l t e r a t i o n o f exchange r a t e . F i r s t , the p o s s i b i l i t y o f the u n m o d i f i e d system i n the slow - 84 -exchange r e g i o n i s e x p l o r e d . (As e x p l a i n e d i n Chapter IV i t i s u n l i k e l y t h a t the system i s i n the f a s t exchange r e g i o n ) . Second, i t w i l l be examined whether i t i s l i k e l y t h a t s u l f o n y l a -t i o n o f the enzyme changes the exchange r a t e from slow t o v e r y  f a s t . Here, the d i s s o c i a t i o n r a t e ( k _ l f o f f r a t e ) f o r the r e a c t i o n E + I £ E I i s r e l a t e d t o the d i s s o c i a t i o n c o n s t a n t (Kj) by the r e l a t i o n , K^ . = k_^/k^, where k^ r e p r e s e n t s the a s -s o c i a t i o n r a t e . I f the a s s o c i a t i o n r a t e s f o r i n h i b i t o r s are 9 assumed t o be those of d i f f u s i o n c o n t r o l l e d r e a c t i o n s * {- 10 M - 1 S , i t i s p o s s i b l e t o c a l c u l a t e the o f f r a t e s from the known d i s s o c i a t i o n c o n s t a n t s . In the p r e s e n t work, the d i s s o c i -—6 —3 a t i o n c o n s t a n t s v a r y from 3.3 x 10 M f o r e s e r i n e t o 5 x 10 M f o r TMA. The e s t i m a t e d o f f r a t e s suggest t h a t i t i s u n l i k e l y f o r any o f the i n h i b i t o r s e x c e p t e s e r i n e t o b i n d t o AchE at a slow exchange r a t e . The p o s s i b i l i t y of changing the exchange r a t e from slow t o v e r y f a s t by s u l f o n y l a t i n g the enzyme may be i n f e r r e d from s t u d i e s comparing the i n h i b i t o r b i n d i n g c o n s t a n t s t o n a t u r a l AchE w i t h those t o v a r i o u s m o d i f i e d AchE. To change from the slow r e g i o n t o the v e r y f a s t r e g i o n , the o f f r a t e ( P B A or k_-^ ) must i n c r e a s e by a p p r o x i m a t e l y 10^ t i m e s . T h i s would r e s u l t i n an i n c r e a s e o f the d i s s o c i a t i o n c o n s t a n t by a t l e a s t the same * T h i s i s a r e a s o n a b l e a s s u m p t i o n , c o n s i d e r i n g the f a c t t h a t the b i n d i n g of both p e r i p h e r a l and a c t i v e s i t e s e l e c t i v e l i g a n d s ( p r o p i d i u m , N - m e t h y l a c r i d i n i u m and l - m e t h y l - 7 - h y d r o x y -q u i n o l i n i u m ) appears t o be d i f f u s i o n c o n t r o l l e d (77,113). - 85 -amount. A c c o r d i n g t o Main (46) , a c y l a t e d AchE indeed g i v e s l a r g e r d i s s o c i a t i o n c o n s t a n t s than n a t u r a l AchE. T h i s i s p a r -t i c u l a r l y t r u e f o r s i t e d i r e c t e d i n h i b i t o r s , such as c h o l i n e and PTA, a l t h o u g h the obser v e d i n c r e a s e i s not g r e a t e r than 50 f o l d . S i m i l a r r e s u l t s on the b i n d i n g o f v a r i o u s i n h i b i t o r s t o c a r b a -m y l a ted AchE were o b t a i n e d from s t u d i e s of the r a t e s of d e c a r -b a m y l a t i o n i n the presence of i n h i b i t o r s ( 51). The b i n d i n g of b i s q u a r t e r n a r y l i g a n d s b e a r i n g b u l k y s u b s t i t u e n t s t o methane-s u l f o n y l a t e d AchE was a l s o i n v e s t i g a t e d , u s i n g s p i n l a b e l s (79) and f l u o r e s c e n c e measurements ( 7 6 ) . Here, a 20 f o l d i n -c r e a s e o f the d i s s o c i a t i o n c o n s t a n t s was found. Only one l i g a n d - 3 - h y d r o x y p h e n y l t r i m e t h y l ammonium (3HPTA) - showed an e x c e p t i o n a l d e c r e a s e i n the b i n d i n g a f f i n i t y t o the methane-s u l f o n y l a t e d AchE. W i l s o n observed a 100 f o l d i n c r e a s e (92, 101), whereas Suszkiw (102) r e p o r t e d the b i n d i n g t o be com-p l e t e l y a b o l i s h e d . These o v e r a l l r e s u l t s seem t o i n d i c a t e t h a t the b i n d i n g of a c t i v e s i t e d i r e c t e d i n h i b i t o r s does not s i g n i -f i c a n t l y d e c r e a s e the b i n d i n g a f f i n i t y and hence the exchange r a t e cannot be a l t e r e d from slow t o v e r y f a s t by m o d i f y i n g the enzyme. The v a l u e f o r m o d i f i e d enzyme, however, must be i n t e r -p r e t e d c a r e f u l l y and the o b s e r v a t i o n o f o n l y a s m a l l change i n the b i n d i n g c o n s t a n t (K^) does not n e c e s s a r i l y mean t h a t the b i n d i n g c o n s t a n t a t the a c t i v e s i t e has h a r d l y changed. Be-cause f o r most l i g a n d s , t h e r e e x i s t s no p r o o f t h a t they don't - 86 -bind to peripheral s i t e s as well; in fact many ligands, es-p e c i a l l y bisquarternary ligands, are known to bind at both types of s i t e . It may then be possible for an inh i b i t o r to bind to a peripheral s i t e when an active s i t e i s not available. In this case, the binding constant for the peripheral s i t e , which could be quite similar to that for an active s i t e , would appear to be Kj for AchE as a whole, while the actual change at the o r i g i n a l binding s i t e might have been much bigger. And indeed for 3-HPTA - the only i n h i b i t o r which the non-binding to p e r i -pheral s i t e s has been conclusively shown for to date - a s i g -n i f i c a n t change in has been found for the interaction with modified AchE. Since from a l l the arguments discussed previously, i t seems unlikely that the observed difference in linewidth can be at-tributed to an a l t e r a t i o n of the exchange rate following methanesulfonylation of the enzyme, we'll now consider the second possible reason, namely the f l e x i b i l i t y of bound i n h i -b i t o r s . It i s hard to imagine that attaching a modifying group to the e s t e r a t i c s i t e of AchE w i l l not s t e r i c a l l y hinder the motion of an i n h i b i t o r bound to the anionic s i t e at the active surface, since these s i t e s are in close proximity. Of these i n h i b i t o r s which bind to the active anionic s i t e , atropine and eserine show a greater value of =1— compared with PTA. This supports our concept since atropine and eserine are larger molecules than PTA. The results with TMA and spiro i n h i b i t o r - 87 -can be e x p l a i n e d s i m i l a r l y . TMA i s a s m a l l m o l e c u l e , and as might be e x p e c t e d , shows no l i n e b r o a d e n i n g , even w i t h m o d i f i e d AchE, whereas the s p i r o i n h i b i t o r as a v e r y b u l k y m o l e c u l e shows b r o a d e n i n g even w i t h n a t u r a l enzyme. F u r t h e r e v i d e n c e f o r the importance of the geometry around the a c t i v e s i t e i s p r o v i d e d by the f o l l o w i n g o b s e r v a t i o n s . ESR s t u d i e s suggest t h a t the a c t i v e s i t e r e g i o n o f n a t u r a l AchE, which i s l a r g e and/or l o c a t e d on the s u r f a c e of the enzyme (109,110), i s more s p a c i o u s than t h a t o f a - c h y m o t r y p s i n and e l a s t a s e . T h i s might e x p l a i n why, u n l i k e o t h e r enzymes (86,103), no b r o a d e n i n g i s o b s e r v e d i n the p r e s e n t e x p e r i m e n t u n l e s s the enzyme i s modi-f i e d . When the m o d i f i c a t i o n of the enzyme was done w i t h methane-s u l f o n y l f l u o r i d e , i t was found t h a t the r e a c t i o n i s a c c e l e r -ated by s m a l l m o l e c u l e s such as TMA, TeMA and TEA (and PTA t o a much l e s s e r e x t e n t ) , whereas l a r g e r m o l e c u l e s , f o r example g a l l a n i n e , tetra-n-butylammonium and tetra-n-propylammonium, i n h i b i t e d s u l f o n y l a t i o n (47,68,69). However the r a t e o f s u l -f o n y l a t i o n by e s t e r s o f m e t h a n e s u l f o n i c a c i d s , i n p a r t i c u l a r those c o n t a i n i n g a q u a t e r n a r y ammonium f u n c t i o n , i s slowed by s m a l l and l a r g e i n h i b i t o r s a l i k e (101). T h i s a l s o i m p l i e s t h a t both TMA and PTA b i n d t o the a n i o n i c s i t e a t the a c t i v e r e g i o n where a l a r g e i n h i b i t o r can s t e r i c a l l y i n t e r f e r e w i t h the e s -t e r a t i c s i t e . I t can t h e r e f o r e be c o n c l u d e d t h a t the observed i n h i b i t o r l i n e w i d t h d i f f e r e n c e between the m o d i f i e d and unmodi-f i e d system i s due t o a change i n f l e x i b i l i t y (hence i n 7 ^ — )r r a t h e r than a change i n the exchange r a t e . (The l a t t e r - 88 -p o s s i b i l i t y , however, can n o t be e n t i r e l y e x c l u d e d . ) T h i s sug-g e s t s , as ESR s t u d i e s d i d , t h a t the a c t i v e r e g i o n o f n a t u r a l AchE i s l a r g e enough t o l e t bound i n h i b i t o r s r o t a t e f r e e l y , the e f f e c t i v e s i z e b e i n g a t l e a s t the s i z e of a t r o p i n e , and more s p a c i o u s than t h a t of the r e l a t e d enzyme, a - c h y m o t r y p s i n , which e x h i b i t e d c o n s i d e r a b l y l a r g e r NMR bound l i n e w i d t h s w i t h i n h i b i -t o r s comparable i n s i z e t o the ones used i n the c u r r e n t e x p e r i -ments (86,112). The bound l i n e w i d t h o f PTA ( 1 = 15 Hz) t o 1 T A2B the m o d i f i e d AchE a l s o i n d i c a t e s the a c t i v e c e n t e r o f the enzyme i s l a r g e enough t o a f f o r d r o t a t i o n a l freedom of a bound PTA m o l e c u l e even when the e s t e r a t i c s i t e i s b l o c k e d w i t h a MeSC^ group. C. POSSIBLE EXTENSION OF STUDIES AchE has l o n g been of r e s e a r c h i n t e r e s t f o r i t s e f f i c i e n t mechanism of h y d r o l y s i s and moreover f o r i t s i n t e r a c t i o n w i t h p h a r m a c o l o g i c a l l y i m p o r t a n t d r u g s . As mentioned e a r l i e r , t h i s enzyme, however, has not been s t u d i e d n e a r l y as e x t e n s i v e l y as the c l o s e l y r e l a t e d s e r i n e p r o t e a s e s , e s p e c i a l l y w i t h s p e c t r o -s c o p i c t e c h n i q u e s , s i n c e s u f f i c i e n t q u a n t i t i e s of h i g h p u r i t y AchE have n o t been a v a i l a b l e u n t i l r e c e n t l y . The r e c e n t s p e c t r o s c o p i c s t u d i e s on AchE (by f l u o r e s c e n c e and ESR) p r o v i d e d d e t a i l e d i n -f o r m a t i o n about t h i s enzyme a t the m o l e c u l a r l e v e l . A l t h o u g h NMR s p e c t r o s c o p y has o n l y been used by one group so f a r , more f r e -quent use i s e x p e c t e d f o r the f u t u r e , s i n c e t h i s t e c h n i q u e i s cap-a b l e o f m o n i t o r i n g the i n t e r a c t i o n o f any l i g a n d w i t h AchE. As - 89 -l a r g e r amounts of pure AchE are now becoming a v a i l a b l e , the poor s i g n a l - t o - n o i s e r a t i o of NMR compared t o the o t h e r s p e c t r o s c o p i c t e c h n i q u e s w i l l become l e s s i m p o r t a n t . G r e a t e r s e n s i t i v i t y i s a l s o a c h i e v e d by m o n i t o r i n g a CH^ group, which i s c o n t a i n e d i n most i n h i b i t o r s and d r u g s . Choosing i n h i b i t o r s w i t h s m a l l d i s -s o c i a t i o n c o n s t a n t s (K^.) w i l l a l s o g i v e f u r t h e r s e n s i t i v i t y by a l a r g e r c o n t r i b u t i o n of s i g n a l s o f bound i n h i b i t o r . However i t cannot be c o n c l u d e d t h a t the s m a l l e r the the more u s e f u l the i n h i b i t o r s , as the m o l e c u l e s w i t h s m a l l e r are more l i k e l y t o undergo exchange a t a s l o w e r r a t e . The drawback of i n s e n s i t i v i t y can a l s o be l a r g e l y overcome by u s i n g NMR s p e c t r o m e t e r s w i t h h i g h and homogeneous magnetic f i e l d s (e.g. s u p e r c o n d u c t i n g magnets) and w i t h h i g h s i g n a l - t o - n o i s e r a t i o . T h i s w i l l make i t pos-s i b l e t o use s m a l l c o n c e n t r a t i o n s o f i n h i b i t o r s g i v i n g b e t t e r r a t i o o f bound t o f r e e i n h i b i t o r ( f D ) . From the e q u a t i o n s i n hi T a b l e I I - l i t i s c l e a r t h a t i n both the slow and v e r y f a s t ex-change r e g i o n the p o s s i b i l i t i e s f o r o b s e r v i n g l i n e b r o a d e n i n g w i l l i n c r e a s e w i t h l a r g e r f„. Hence, i t may w e l l be p o s s i b l e t o observe a b r o a d e n i n g of the NMR peaks i n the i n t e r a c t i o n of i n h i b i t o r s w i t h the n a t u r a l enzyme, i f a s m a l l e r i n h i b i t o r con-c e n t r a t i o n c o u l d be used, as would be the case w i t h a 270 MHz s p e c t r o m e t e r . Once any l i n e b r o a d e n i n g i s observed w i t h n a t u r a l enzyme, the exchange r e g i o n can be d e t e r m i n e d , r e s o l v i n g the q u e s t i o n d i s c u s s e d e a r l i e r , as t o whether the m e t h a n e s u l f o n y l a -t i o n has changed e i t h e r the exchange r a t e s or the f l e x i b i l i t y of bound i n h i b i t o r s . - 90 -A l s o , i f the bound l i n e w i d t h of an i n h i b i t o r i n t e r a c t i n g w i t h the n a t u r a l enzyme c o u l d be o b t a i n e d , NMR s p e c t r o s c o p y would p r o v i d e a u s e f u l t o o l t o i n v e s t i g a t e l i g a n d - i n d u c e d con-f o r m a t i o n a l changes i n AchE. There have been many ex p e r i m e n t s which suggested such a change b e i n g a s s o c i a t e d w i t h a l i g a n d b i n d i n g t o AchE. For i n s t a n c e , i t i s w i d e l y a c c e p t e d t h a t the i n f l u e n c e o f q u a t e r n a r y i n h i b i t o r s on the enzyme a c t i v i t y i s brought about by a s s o c i a t i o n t o a p e r i p h e r a l s i t e and a conco-m i t a n t c o n f o r m a t i o n a l change o f AchE (21,69,104). S i m i l a r con-c l u s i o n s have been reached by s t u d y i n g the a c y l a t i o n of the en-zyme by i r r e v e r s i b l e i n h i b i t o r s , such as carbamates and e s t e r s of p h o s p h o r i c and s u l f o n i c a c i d s i n the presence of r e v e r s i b l e i n h i b i t o r s (47,51). B o l g e r and T a y l o r a l s o proposed a c o n f o r -m a t i o n a l change o f the enzyme from t h e i r f l u o r e s c e n c e s t u d y i n which t h e y o b s e r v e d a s h i f t o f the e m i s s i o n maximum o f p r o t e i n f l u o r e s c e n c e upon l i g a n d c o m p l e x a t i o n w i t h AchE and a slow u n i -m o l e c u l a r i n t e r c o n v e r s i o n of the enzyme s p e c i e s (66). Most o f the e x p e r i m e n t a l r e s u l t s s u g g e s t i n g a c o n f o r m a t i o n a l change of the enzyme, however, f a i l t o p r o v i d e d i r e c t e v i d e n c e . The f i r s t c o n c l u s i v e e v i d e n c e f o r l i g a n d - i n d u c e d c o n f o r m a t i o n a l change of AchE i s g i v e n i n a r e c e n t r e p o r t by E p s t e i n e t a l (105). In t h e i r s t u d y f l u o r e s c e n t phosphonates which c o n j u g a t e w i t h the s e r i n e m o i e t y a t the a c t i v e - c e n t e r are employed to demonstrate d i r e c t l y t h a t p e r i p h e r a l s i t e d i r e c t e d l i g a n d s a l t e r the a c t i v e s i t e c o n f o r m a t i o n . The NMR l i n e w i d t h o f a bound i n h i b i t o r i s v e r y s e n s i t i v e t o the s u r r o u n d i n g environment and i t a l s o s h o u l d - 91 -provide information about a conformational change of the enzyme near the binding s i t e . Such an experiment can be designed by 1. modifying the peripheral s i t e by a s i t e s p e c i f i c reagent (57,106) and measuring -=r— of a ligand associating with the active s i t e , or 2. l e t t i n g a ligand, which associates only with the active center, interact with AchE in the presence and absence of a peripheral s i t e directed ligand and comparing the value ~ — values of the active s i t e directed ligand. 12B As mentioned in Chapter I I , binding modes for most i n h i b i t o r s are unfortunately not c l e a r l y elucidated yet, espe c i a l l y since very l i t t l e i s known so far about the extent of their binding to secondary s i t e s . The choice of i n h i b i t o r s for the above described experiments i s thus r e s t r i c t e d to compounds such as edrophonium and propidium, which bind exclusively to the active and peripheral s i t e s , respectively. The conformational change of the enzyme can also be studied by modifying the active s i t e of the enzyme with acid transfering i r r e v e r s i b l e i n h i b i t o r s . An example of the investigation of the active s i t e area with a NMR technique using an acid transfering i n h i b i t o r i s that of that by Reech et a l , in which diisopropylfluorophosphate d e r i -vatives of chymotrypsin and chymotrypsinogen are compared by phosphorus-31 NMR (111). In our case, d i r e c t observation of i n h i b i t o r s covalently bound to the e s t e r a t i c s i t e with NMR spectroscopy in the presence and absence of other ligands might elucidate the possible a l l o s t e r i c e f f e c t of ligands binding to - 92 -the p e r i p h e r a l s i t e . The advantage of u s i n g NMR s p e c t r o s c o p y here i s t h a t the change i n the l i n e w i d t h of a bound i n h i b i t o r , u n l i k e the change i n f l u o r e s c e n c e , p r o v i d e s a measure of the ex-t e n t of the c o n f o r m a t i o n a l change. I t i s p o s s i b l e t o g a i n more d e t a i l e d i n f o r m a t i o n c o n c e r n i n g the n a t u r e of the c o n f o r m a t i o n a l change of AchE by d e t e r m i n i n g TJT1— f o r v a r i o u s r e g i o n s o f an i n -i2B h i b i t o r m o l e c u l e . T h i s type of p r e c i s e m o l e c u l a r l e v e l under-s t a n d i n g o f the i n f l u e n c e o f l i g a n d b i n d i n g on AchE w i l l be r e q u i r e d i n answering a l o n g sought q u e s t i o n : why c e r t a i n p e r i -p h e r a l l i g a n d s a c c e l e r a t e s u b s t r a t e (or i r r e v e r s i b l e i n h i b i t o r ) r e a c t i o n w h i l e the o t h e r d e c e l e r a t e . The a b i l i t y t o m o nitor the b i n d i n g a t d i f f e r e n t groups w i t h i n the i n h i b i t o r m o l e c u l e s e p a r a t e l y w i l l a s s i s t i n i d e n t i f y i n g p o s s i b l e m u l t i p l e b i n d i n g s i t e s near the a c t i v e r e g i o n , proposed by two workers (46,107), and c h a r a c t e r i z i n g them. For i n s t a n c e , the response of the l i n e w i d t h (-^ -) o f both a t r o p i n e p h e n y l and N-methyl peaks (and i 2 a l s o o f t r o p i n e and t r o p i c a c i d , the two s u b u n i t s of the a t r o -p i n e m o l e c u l e , c o n t a i n i n g e i t h e r charged m e t h y l group or the p h e n y l r i n g ) c o u l d be s t u d i e d under the i n f l u e n c e of d i f f e r e n t p.H., i o n i c s t r e n g t h and o t h e r r e v e r s i b l e i n h i b i t o r s . A l t h o u g h T=—-— p r o v i d e s a u s e f u l measure i n the a n a l y s i s of A2B i n h i b i t o r i n t e r a c t i o n s w i t h AchE a t the m o l e c u l a r l e v e l , i t i s o b t a i n a b l e o n l y when the system i s i n the v e r y f a s t exchange r e g i o n . A s t u d y of under the exchange c o n d i t i o n s , which 1 1 p r o v i d e s b a s i c a l l y the same i n f o r m a t i o n as t h a t o f 7=7— (so shown l2 i n T a ble I I - l ) , i s much l e s s f r e q u e n t l y performed s i n c e the - 93 -measurements are more time consuming. =^ -f however, can be 1 1 useful when the exchange rate i s not fast enough and = — cannot i2B be obtained. The exchange rate i s more l i k e l y to be very fast in T^ time scale than in T 2 time scale because i s indepen-dent of Ato (as seen in Table I I - l ) and T^ i s generally larger than T 0. Hence i t i s s t i l l possible to calculate 7=^—,. which ^ i l B provides the same type of information as — — , while the system , X2B is too slow to calculate 7=— and such a case i s indeed reported l2B by Sykes (103). - 94 -GLOSSARY OF TERMS AND ABBREVIATIONS Ach: a c e t y l c h o l i n e AchE: a c e t y l c h o l i n e s t e r a s e a l l o s t e r i c s i t e : a b i n d i n g s i t e on an enzyme where a l i g a n d can b i n d and cause a s t r u c t u r a l change i n the enzyme m o l e c u l e ESR: e l e c t r o n s p i n resonance e s t e r a t i c s i t e : a s i t e c o n s i s t i n g of the r e s i d u e s which d i r e c t l y p a r t i c i p a t e i n the making and b r e a k i n g o f bonds HPTA: 3-hydroxyphenyltrimethylammonium i o n NMR: n u c l e a r magnetic resonance p r o t e o l y s i s : enzymic h y d r o l y s i s of p e p t i d e bonds PTA: phenyltrimethylammonium i o n s e r i n e p r o t e a s e : an enzyme which can s p l i t c e r t a i n p e p t i d e l i n k a g e s a t any p o i n t i n a p e p t i d e c h a i n and have a r e a c t i v e s e r i n e r e s i d u e TeMA: tetramethylammonium i o n TMA: trimethylammonium i o n - 95 -REFERENCES 1. 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