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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 U n i v e r s i t y , 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  t h i s t h e s i s as conforming  to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA April  1980  (g) Junko Maetani C a r r u t h e r s ,  1980  In p r e s e n t i n g  this  an a d v a n c e d  degree  the  shall  I  Library  further  for  agree  thesis at  freely  of  extensive  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  this  written  thesis for  by t h e  i s understood  f i n a n c i a l gain shall  permission.  Department of  rJMb/wuV^  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  n„tP  It  <kjJr S i  Columbia  hyp  of  the  requirements  B r i t i s h Columbia,  available for  permission for  by h i s r e p r e s e n t a t i v e s . of  fulfilment  the U n i v e r s i t y  make i t  that  in partial  r e f e r e n c e and copying of  this  copying or  that  study. thesis  Head o f my D e p a r t m e n t  that  not  I agree  for  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT  Acetylcholinesterase part  in neural  i s an enzyme w h i c h p l a y s an  transmission.  T h i s enzyme, h o w e v e r , has  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 t h e level,  due  acterized for  t o the d i f f i c u l t i e s forms of  preparing  and  various  t h e enzyme.  i n o b t a i n i n g p u r e and This  s t u d i e s on  inhibitors.  the  well  thesis describes  interaction  ly  reported  ammonium g r o u p .  large line  inhibitor  upon i n t e r a c t i o n w i t h n a t u r a l A c h E , t h e p r e s e n t no  significant  hibitors  line  i n the  investigate  the  b r o a d e n i n g w i t h any  same c o n c e n t r a t i o n binding  site  zyme, t h e a c t i v e e s t e r a t i c sulfonylation. indeed  show l i n e  i n the  s i t e was  cause of  c h a n g e s i n e x c h a n g e r a t e s and The  present  of the i n to further  of the  en-  limit.  l i n e w i d t h s of  flexibility the  by AchE  t o bound l i n e w i d t h s  AchE i s d i s c u s s e d  overall results confirm  showed  this modified  theoretical  the d i f f e r e n c e s i n o b s e r v e d modified  results  permanently blocked  broadenings, g i v i n g r i s e  b e t w e e n n a t u r a l and  previousresonances  In o r d e r  I n h i b i t o r s interacting with  which are w e l l w i t h i n the e x p e c t e d  NMR  active center  small  but a l l  e x c e p t one  range.  form  from a  In c o n t r a s t t o  broadenings of  a method  between t h i s  I n h i b i t o r s range i n s i z e  char-  electr icus,  trimethylammonium ion to a l a r g e a t r o p i n e molecule, contain a quaternary  not  molecular  s u c h a f o r m o f AchE f r o m E l e c t r o p h o r u s  f o l l o w e d by NMR  important  The  inhibitors  i n terms of  o f bound  inhibitors.  importance of  prior  c h a r a c t e r i z a t i o n o f t h e enzyme hence e x p l a i n i n g between the l i n e  broadenings of i n h i b i t o r s i n t e r a c t i n g with  n a t u r a l A c h E o b s e r v e d by a n o t h e r w o r k e r current  w o r k , and  a l AchE i s l i k e l y the  also  indicate that  and o n e s o b s e r v e d i n  the a c t i v e r e g i o n  of  natur-  t o be l a r g e , t h e 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  s i z e of a t r o p i n e ,  zyme .  the d i s c r e p a n c y  and/or  located  on t h e s u r f a c e  of the  en-  - iv -  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF FIGURES AND TABLES ACKNOWLEDGEMENT CHAPTER I :  v i i ix  INTRODUCTION  1  A.  Acetylcholinesterase  1  B.  E x t r a c t i o n and P u r i f i c a t i o n  4  C.  Structure  6  i) ii) D.  ii)  Substrate  Reaction  I n h i b i t o r s and Their I n t e r a c t i o n with the Enzyme  S p e c t r o s c o p i c Techniques i n the Study of the C a t i o n i c Mechanism of A c e t y l c h o l i n e s t e r a s e  CHAPTER I I : A.  The 11S Species  C a t a l y t i c Mechanism i)  E.  The 9S, 14S and 18S Species  THEORETICAL BACKGROUND  7 11 12 13 15 20 25  The Lineshape A n a l y s i s f o r a Molecule Undergoing Chemical Exchange when the P o p u l a t i o n of the Two S i t e s are G r e a t l y D i f f e r e n t  25  B.  Expected Linewidth i n NMR Spectra f o r a Methyl Group Attached t o a Macromolecule  32  C.  P l o t s t o Obtain  34  CHAPTER I I I : A.  Bound Linewidth  EXPERIMENTAL METHODS  P u r i f i c 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 i)  E x t r a c 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 from E l e c t r i c E e l i n High S a l t Media  36 36  -  V  -  Page ii)  Purification  iii) B.  37  a)  Preparation  37  b)  R u n n i n g o f t h e Column  o f t h e Column  Trypsin  39  Assay  Conversion of Acetylcholinesterase  ii)  by  A f f i n i t y Chromatography  Acetylcholinesterase  i)  C.  of Acetylcholinesterase  41 t o 11S Form  Digestion  C h a r a c t e r i z a t i o n of Converted cholinesterase  Acetyl-  46  C o n c e n t r a t i o n o f t h e Enzyme w i t h t h e Amicon F i l t e r  46  Other methods f o r C o n c e n t r a t i n g D 0 Buffer  47  AchE i n t o  2  D.  Preparation of Modified Verification i) ii)  Preparation  Enzyme and i t s  48  of Eseroline  48  S u l f o n y l a t i o n of Acetylcholinesterase  50  E.  Stability  F.  NMR M e a s u r e m e n t s  CHAPTER I V :  43  Samples  2  ii)  42 42  Preparation of Acetylcholinesterase in D 0 Buffer i)  ..  o f t h e Enzyme  51 52  RESULTS  54  A.  Stability  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.  Eserine  64  D.  I n h i b i t o r s with Methanesulfonylated cholinesterase  CHAPTER V: A.  and P u r i t y o f t h e Enzyme  54  w i t h C a r b a m y l a t e d Enzyme  DISCUSSION  Inhibitors with  Acetyl-  71 79  U n m o d i f i e d AchE  79  - vi -  Page  B.  I n h i b i t o r s with M o d i f i e d Enzyme  83  C.  P o s s i b l e E x t e n t i o n of S t u d i e s  88  GLOSSARY  94  REFERENCES  95  -  v i i  -  L I S T OF FIGURES AND  TABLES  Figure  Page  I-l  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  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 E. E l e c t r i c u s  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  Acetylcholine  16  Structure  III-3 IV-1 IV-2  IV-3 IV-4 IV-5 IV-6  of  Sites of Acetylcholinesterase  1-5  III-2  9  14  Binding  III-l  from  by A c e t y l c h o l i n e s t e r a s e  1-4  II-l  8  o f AchE I n h i b i t o r s  18  A P l o t o f 1/T 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 : An E l u t i o n 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 f r o m an A f f i n i t y Column 2  33 40  Sucrose Gradient 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 b e f o r e and a f t e r t h e T r e a t m e n t w i t h T r y p s i n  45  An Enzyme D i s t r i b u t i o n i n a Tube a f t e r Centrifugation  49  S u c r o s e G r a d i e n t P r o f i l e o f 11S AchE b e f o r e a f t e r t h e S t o r a g e i n 4M N a C l B u f f e r a t 4°C  and 55  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 11S 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 Conditions  55  Structure of Acetylcholinesterase u s e d i n NMR E x p e r i m e n t  57  The H NMR S p e c t r a U n m o d i f i e d AchE  of Atropine  The tt NMR S p e c t r a C a r b a m y l a t e d AchE  of Eserine  X  1  Inhibitors  Sulfate  with 59  Sulfate  with 65  A P l o t o f AAy f o r t h e C - m e t h y l P r o t o n of E s e r i n e with respect t o / l ~ E  K  0  0  D  Resonances 68  - viii  -  Page  A P l o t o f R e c i p r o c a l o f AAy f o r the C - m e t h y l Resonances of E s e r i n e v s . V a r y i n g Concentrations of Eserine 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 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 M o d i f i e d AchE  IV-7  IV-10 IV-11  of A t r o p i n e  Hydro-  Sulphate  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 1 w i t h M o d i f i e d AchE .  with 73 Chloride  A P l o t o f the R e c i p r o c a l o f AAy 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 Concentrations of A t r o p i n e S u l f a t e  74  76  IV-12  A P l o t o f AAy 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 AAy f o r the M e t h y l Group Resonances of P h e n y l t r i m e t h y l a m m o n i u m v s . Concentration  77  E x p e c t e d R e l a x a t i o n Times i n P r e s e n c e of the C h e m i c a l E x c h a n g e when A i s D o m i n a n t S p e c i e s i n Solution  30  O b s e r v e d L i n e B r o a d e n i n g s o f NMR R e s o n a n c e s 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  Table II-l  IV-1  -  ix  -  ACKNOWLEDGEMENT  Although  I would l i k e  d u r i n g my s t a y to express First,  department,  G.  physical  Webb f o r h i s g e n e r o u s  t o e x t e n d my g r a t i t u d e  Luoma, J .  S m i t h a n d L.  support.  encountered I would  t o my s u p e r v i s o r ,  g u i d a n c e and p o s i t i v e  like  h e l p and u s e f u l  Dr.  encouragement  I would p a r t i c u l a r l y l i k e  w h i c h t h i s work w o u l d n o t have g o t t e n  happy  here  I  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 :  the course o f t h i s work.  Dr.  without  G.  special  Chemistry  Marshall, for his continual  throughout  also  i n the U.B.C.  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  A l a n G.  thank  t o t h a n k many o f t h e p e o p l e  to  suggestions,  o f f the ground.  t o my f r i e n d s W. A p p e l ,  W a l s h , who p r o v i d e d e m o t i o n a l  P.  I am Burns,  as w e l l as  - 1 -  CHAPTER I INTRODUCTION  The work p r e s e n t e d  in this  thesis describes  an i n v e s t i g a -  t i o n o f t h e 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 of  (AchE) and i t s i n h i b i t o r s u s i n g  techniques.  In s p i t e  t h e 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 t h e  functioning of  NMR  o f n e r v e and m u s c l e c e l l s ,  the d e t a i l e d  function  t h i s enzyme i s a s y e t n o t w e l l u n d e r s t o o d c o m p a r e d w i t h  other  serine  purifying Until  e n z y m e s , b e c a u s e a s t a n d a r d way o f i s o l a t i n g and  t h i s enzyme was n o t e s t a b l i s h e d  recently,  f o r a long  i t has a l s o n o t been p o s s i b l e  f i c i e n t q u a n t i t i e s of high enzyme a t t h e m o l e c u l a r  time.  to obtain  suf-  p u r i t y enzymes f o r a n a l y s i s o f  level.  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 o f t h e r o l e o f AchE i n n e u r a l for  isolation  transmission,  followed  and t h e s t r u c t u r e d e t e r m i n a t i o n  by t h e t e c h n i q u e o f t h e enzyme.  The c a t a l y t i c m e c h a n i s m o f t h e enzyme and t h e p r e s e n t  knowledge  of  i t s interaction with  followed  by  a discussion  inhibitor  i s then c o n s i d e r e d ,  o f t h e methods a v a i l a b l e t o i n v e s t i g a t e t h e  d y n a m i c a s p e c t s o f t h e enzyme. A.  ACETYLCHOLINESTERASE Acetylcholinesterase  i s t h e enzyme w h i c h c a t a l y z e s t h e  h y d r o l y s i s of a c e t y l c h o l i n e excitable  membranes.  and i s w i d e l y  distributed in  - 2 -  0 C — O — C H C H — N (CH )  CH  2  2  3  + AchE -> CH-.COOH + H O — C H C H „ — N ( C H ^ ) H~0 0  3  acetylcholine  choline  I n e a r l y s t u d i e s , t h e enzyme c a t a l y z i n g t h e h y d r p l y s e s c h o l i n e e s t e r s was f o u n d a s s o c i a t e d tissue  (1,2).  with  of  n e r v e and m u s c l e  L a t e r , enzymes h a v i n g t h e same f u n c t i o n were  found  i n many o t h e r  tissues.  tinct  t y p e s o f enzyme i n b l o o d  other  i n t h e serum - t h e k i n e t i c p r o p e r t i e s o f t h e s e two  enzymes became t h e b a s i s tissues. thesis,  A f t e r the discovery  enzymes i n o t h e r  A c e t y l c h o l i n e s t e r a s e , t h e enzyme s t u d i e d i s the red c e l l  type which hydrolyzes  Acetylcholinesterase species  catalyze  (3) - one i n t h e r e d c e l l s , t h e  forclassifying  more r a p i d l y t h a n b u t y l c h o l i n e  all  of animals.  i n contrast  i s found  in this  acetylcholine far  t o t h e serum  type.  i n the nervous t i s s u e s o f  I t s most i m p o r t a n t f u n c t i o n  i s to  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 impulse.  Acetylcholine  c h e m i c a l t r a n s m i t t e r s w h i c h makes i t p o s s i b l e impulses across nerve c e l l s , is  o f two d i s -  synapses, a s p e c i f i c  or a nerve c e l l  i s one o f t h e to transmit  s t r u c t u r e b e t w e e n two  and a m u s c l e c e l l .  The f o l l o w i n g  a schematic diagram o f a synapse.  synaptic  vesicles  Acetylcholinesterase  axon  postsynapti c membrane  synaptic cleft  presynapti membrane direction  of nerve  nerve  impulse  - 3 -  Within  t h e b u l b o u s s t r u c t u r e a t t h e end o f t h e p r e s y n a p t i c  axon, there  a r e many s m a l l v e s i c l e s e a c h c o n t a i n i n g i n t h e 4  order  o f 10  molecules of the neurotransmitter.  ergic  synapse t h i s n e u r o t r a n s m i t t e r  a r r i v a l of a nerve the  impulse  At the c h o l i n -  i s a c e t y l c h o l i n e and t h e  a t t h e p r e s y n a p t i c membrane  causes  l i b e r a t i o n o f quanta of a c e t y l c h o l i n e molecules i n t o the  synaptic c l e f t . synaptic c l e f t specific  The a c e t y l c h o l i n e t h e n d i f f u s e s a c r o s s t h e to the  receptor  acetylcholine  p o s t s y n a p t i c membrane, where i t b i n d s t o  molecules.  These r e c e p t o r m o l e c u l e s ,  receptors, are associated with  the  ion channels.  On  t h e b i n d i n g o f a c e t y l c h o l i n e , t h e c h a n n e l s open and d e p o l a r i z e t h e 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 i n w a r d of N a .  This d e p o l a r i z a t i o n of the p o s t s y n a p t i c  +  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 t h e a d j a c e n t membrane.  current  membrane  axon o r muscle  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 quickly  t o r e s t o r e t h e e x c i t a b i l i t y o f t h e p o s t s y n a p t i c membrane. i s achieved  This  by t h e 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 concentrated  at the surface of the postsynaptic  membrane. Acetylcholinesterase prepared  (AchE) u s e d  i n t h i s e x p e r i m e n t was  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 o f c e l l s called  e l e c t r o p l a x e s , w h i c h have e v o l v e d  They r e t a i n t h e e x c i t a b l e o u t e r lost  the c o n t r a c t i l e  apparatus.  from muscle  cells.  membrane o f m u s c l e b u t have The e l e c t r i c  e e l i s very  - 4 -  rich of  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%  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  reason,  t h e 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 large q u a n t i t i e s of  AchE.  s o u r c e o f AchE a r e t h e e l e c -  tric  Although organs  t h e most p o p u l a r  o f t h e e l e c t r i c e e l and t h e t o r p e d o , o t h e r  sources  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 h e a d and human b r a i n .  (It  is  being  t h u s e v i d e n t t h a t AchE may o c c u r  a s s o c i a t e d w i t h nerves.) may  show a v a r i a t i o n  bitors  i n tissues without  However, AchE from d i f f e r e n t  in reactivity  toward  irreversible  ( 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  bovine e r y t h r o c y t e AchE, but does n o t i n h i b i t r a t e ) AchE from t h e e l e c t r i c reported differences, s h o u l d be a p p l i c a b l e the overwhelming  (5,6).  (at a  inhiinhibits  measurable  In s p i t e of these  t h e f i n d i n g s f o r A c h E f r o m one  source  t o t h e enzymes f r o m o t h e r s o u r c e s , due t o  similarities  s t r a t e and i n h i b i t o r  organ  sources  i n t h e enzyme s t r u c t u r e , t h e s u b -  specificity,  and t h e t u r n o v e r numbers o f  acetylcholine (7). B.  EXTRACTION AND  PURIFICATION  The a p p e a r a n c e o f a h e t e r o g e n e o u s AchE i n h i g h i o n i c first  size d i s t r i b u t i o n of  strength extracts of fresh e e l tissue  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  ized the molecular coefficient)  forms o f AchE  was  ( 8 ) . They c h a r a c t e r -  (by t h e i r  sedimentation  t o be t h e 9S, 14S and 18S s p e c i e s .  Other  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 t h e l i s  workers form  - 5 -  (9,10).  I t i s now s p e c u l a t e d  created  a t the synapses  f r o m s m a l l e r b u i l d i n g b l o c k s o f t h e 9S  f o r m o f A c h E , and a l l t h r e e the  t h a t t h e 18S f o r m o f AchE i s  f o r m s , 9S, 14S and 18S c o e x i s t a t  s u r f a c e o f t h e p o s t s y n a p t i c membrane  known t h a t p r o t e o l y s i s d u r i n g create  smaller  o f AchE  (11). I t i s a l s o  the s o l u b i l i z a t i o n  process can  f o r m s o f 9S, 11S and 14S f r o m t h e 18S f o r m  (11,12) .  However, t h e i m p o r t a n c e o f t h e e x t r a c t i o n p r o c e d u r e t o the  subsequent  recognized  c h a r a c t e r i z a t i o n of the i s o l a t e d  until  a b o u t 1970  s i t y o f t h e AchE s p e c i e s organs  (10,14-16).  l e d t o the d i v e r -  t h a t were i s o l a t e d  from  electric  H i s t o r i c a l l y two t y p e s o f e x t r a c t i o n p r o -  c e d u r e have been u s e d eel  ( 1 3 ) , and t h i s  enzyme was n o t  f o r the s o l u b i l i z a t i o n  o f t h e enzyme  e l e c t r i c o r g a n , w h i c h p r o d u c e d w e l l d e f i n e d AchE  tions.  The f i r s t  proteolytic yields  from  distribu-  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 t h e enzyme t o a  agent, or immersing  t h e l i s f o r m o f AchE  the e e l t i s s u e i n toluene;  this  (7). In the second p r o c e d u r e , t h e  electric  organ  produces  a m i x t u r e o f 9S, 14S and 18S f o r m s o f AchE as m e n t i o n e d  previously.  i s homogenized a t h i g h  A f f i n i t y chromatography  ionic  strength,  d e v e l o p e d 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  operation  takes advantage  organic  (11,17).  inhibitors  of these i n h i b i t o r on a r e s i n  matrix.  This  which bind  which  to specific  one-step  o f t h e range o f s m a l l s i t e s o f AchE.  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 When a c r u d e enzyme s o l u t i o n  One  immobilized  permeates  -  through r e s i n matrix,  6  -  the immobilized  l i g a n d s i n t e r a c t with the  enzyme and c a u s e s a p r e f e r e n t i a l r e t a r d a t i o n , t h e b i n d i n g affinity  f o r l i g a n d s t o t h e enzyme v a r y i n g g r e a t l y w i t h t h e i o n i c  s t r e n g t h o f t h e medium. extracted  i n a high  second p r o c e d u r e . matrix  The enzyme u s e d i n t h i s  ionic  with the N-methylacridinium  high  ionic  C.  STRUCTURE  strength  t i s s u e of the e l e c t r i c ferent molecular  i n v o l v e d an  i o n (see F i g u r e  of binding  t o the affinity  1-5) a s a  t o t h e enzyme e v e n a t a  (17,19).  Acetylcholinesterase  ionic  s t r e n g t h medium, a c c o r d i n g  Subsequent procedure  l i g a n d , which i s capable  t h e s i s was  isolated  from the e l e c t r i c  e e l c a n be o b t a i n e d  forms.  organ  i n a number o f d i f -  T h r e e f o r m s c a n be e x t r a c t e d a t h i g h  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  coeffi-  c i e n t s o f 1 8 S , 14S and 9 S , and a l l a p p e a r h i g h l y a s y m m e t r i c (20).  These s p e c i e s a g g r e g a t e a t low i o n i c  2 1 ) , and d e t a i l e d s t u d i e s o f t h e s e only after appropriate  ( 1 1 , 18,  s p e c i e s were t h u s p o s s i b l e  the i n t r o d u c t i o n of a f f i n i t y chromatography with the ligands for purification  at high  The 11S s p e c i e s , h o w e v e r , a d e g r a d a t i o n o f t h e n a t i v e enzyme ionic  strength  strengths  product  strength.  from p r o t e o l y s i s  (8,11), does n o t aggregate even a t v e r y low  (18,22).  f o r e , was a c h i e v e d  ionic  Purification  much e a r l i e r  of t h i s  species  there-  t h a n o f t h e 9S, 14S and 18S  - 7 -  forms,  and  consequently  studied in greater d e t a i l .  f o r m o f t h e enzyme c a n  be p r o d u c e d e i t h e r  o l y s i s d u r i n g s t o r a g e o r by t r e a t i n g proteases. (23)  Silman  other physicochemical o b s e r v a t i o n s on model i s v e r y (24) , e x c e p t is  data  similar  t o t h e one  t h a t the t a i l  ing  (25,26).  proposed e a r l i e r  i s expressed  by  as a t r i p l e  illustrate  the d i f f e r e n t  Rosenberry helix,  and  isolated  i n the presence  s p e c i e s o f AchE  by h o m o g e n i z a t i o n  o f 1-2  M N a C l and  of e e l  the c h e l a t -  The  9S,  14S  and  18S  Species  Three forms o f n a t i v e AchE w i t h s e d i m e n t a t i o n of approximately  9S,  14S  asymmetric p a r t i c l e s of a l l three forms, a tail,  and  18S,  (20,28).  tail  The  i s seen under the e l e c t r o n m i c r o s c o p e are  connected  (25,26),  identified.  While  and the  t h e same l e n g t h , t h e h e a d s o f 9S,  f o r m s c o n t a i n one,  subunits respectively  as  unique elongated s t r u c t u r e  i n w h i c h a m u l t i - s u b u n i t head i s  i s of approximately 18S  coefficients  behave h y d r o d y n a m i c a l l y  t h e s p e c i e s p i c t u r e i n F i g u r e 1-2  and  This  agent.  i)  to  and  S i m p l i f i e d models of m u l t i p l e forms  which are t y p i c a l l y tissue  e e l AchE  together with e l e c t r o n microscopic  t h e a s y m m e t r i c f o r m s o f AchE  o f AchE i n F i g u r e 1-2  electric  m o d e l o f 18S  s t u d i e s of the p r o t e o l y t i c cleavage  shown i n F i g u r e I - l .  (27),  by e n d o g e n o u s p r o t e -  extracts with various  d e s c r i b e d a schematic  b a s e d on d e t a i l e d  lis  The  (29).  the g l o b u l a r form i s o l a t e d  two  and  The  three tetramers  individual  following  of  14S  catalytic  tetramer, which i s  proteolytic  digestion,  -  l  8  -  Q;!t J V^-Q (  O-O/ s s  1  trypsin  J  -S-Scollagenase  > -S-S-  Figure  1-1:  Model Points  of of  the  18S  acetylcholinesterase.  cleavage  by s o d i u m d o d e c y l  and c o l l a g e n a s e a r e shown  by  arrows.  sulfate,  trypsin  - 9 form  sedimentation  molecular  coefficient  weight (total)  OO OO OO \ OO oov/oo  18.4S  oo OO OOVDO  OO QO  $3  Figure  1-2:  i , i 50 ooo  157,000  5  14.2  S  796,000  134,000  9.2  S  410,000  79,000  11.8S  Multiple The  (tail)  forms  3 3 1  000  o f A c e t y l c h o l i n e s t e r a s e from  best determined  values  for  and m o l e c u l a r w e i g h t  tails  a r e shown as t h e d i f f e r e n c e t h e MM o f  are shown.  the  E.Electricus.  both sedumentation  cient  a n d t h e sum o f  >  coeffi-  Molecular weight  between  tetrameric  the t o t a l  unit  MW  present.  of  - 10  the symmetric form,  i s known as t h e l i s  p r o p e r t i e s of d i f f e r e n t the aggregated as  i s their  form,  18S  -  species.  catalytic  s p e c i e s of AchE, w i t h the e x c e p t i o n  f o r m s a p p e a r t o be e s s e n t i a l l y  behavior  The  towards i n h i b i t o r s  AchE, w i t h a m o l e c u l a r  1,150,000 c o n t a i n s 12 c a t a l y t i c  identical  (31).  weight  of  subunits  The  of  (29,30),  principal  approximately  i n i t s head  arranged o  in  three t e t r a m e r i c groups,  and  has  a tail  approximately  500  A  l o n g . E a c h o f t h e t h r e e s u b u n i t t e t r a m e t e r s , as s e e n f r o m F i g u r e I - l , is  l i n k e d by  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  linked  a d i s u l f i d e bond t o one  t o t h e t a i l may  be d e t a c h e d 18S  strand of a t r i p l e  by s o d i u m d o d e n y l  (23,24).  T r y p s i n c l e a v e s the  f o r m o f AchE t o  tetramers  by a t t a c k i n g t h e n e c k o f t h e t r i p l e  o t h e r h a n d , c o l l a g e n a s e p r o d u c e s a 20S heads w i t h a r e s i d u a l attacking  tail  the midpoint  form,  of the t r i p l e  of these conversions d e s t r o y s  helix  14S  (32).  tailed  forms a t low  forms of e l e c t r i c  considered  t o be  34).  i s suggested  This  molecular  ionic  strength  This rod-like  collagen.  by  Either  Since only  by e l e c t r o n m i c r o s c o p y  (34,35).  to  together,  f o r m as w e l l as  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  i n which the  bundles  of  the  the  o r g a n AchE a r e a g g r e g a t e d , t h e t a i l  assemblies  in  consisting  the  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 18S  9S  On  (23,32,33).  s e l f - a s s o c i a t i o n , which i s seen i n the and  sulfate  discrete  helix.  h o l d i n g the t e t r a m e r s  helix.  tails tail  property  of the  is (32,  multi-  are packed s i d e - t o - s i d e i s now  This s u p p o s i t i o n i s supported  by:  considered  akin  1., t h e s i m i l a r i t y  - 11 -  in  amino a c i d c o m p o s i t i o n  ( 2 4 ) , 2., t h e t h r e e - s t r a n d e d  ance a s s e e n i n e l e c t r o n m i c r o s c o p y  ( 2 6 ) , and 3., t h e  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 . close s i m i l a r i t y i n composition branes.  This  appearcharac-  T h e r e i s an o b s e r v e d  of the t a i l  and basement mem-  suggests that the c o l l a g e n - l i k e t a i l  i s derived  f r o m b a s e m e n t membrane c o l l a g e n w i t h i n t h e s y n a p t i c g a p , and functions ii)  i n t h e i m m o b i l i z a t i o n o f t h e enzyme  The 11S  Species  As d i s c u s s e d various  proteases  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  ( F i g u r e 1-2) .  form, a g l o b u l a r  This degradation  tains  The 11S s p e c i e s  f o u r monomers,  molecular  ligand  a 80,000 d a l t o n  s l i g h t a s y m m e t r y ( 3 8 ) . When a  on t h e t e t r a m e r i c  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  catalytically  a c t i v e monomers show  14S and 11S enzyme  (37,38).  f o r e , c a n be c o n s i d e r e d with  respect  with  identical  t h e enzyme, t h e s t o i c h i o m e t r y subunit  and c o n -  subunits,  ( 3 7 ) , w h i c h a p p e a r t o be  but assembled w i t h  interacts with  to give the  so formed l a c k s a t a i l  the s m a l l e s t c a t a l y t i c  w e i g h t 80,000  structurally  with  f r o m t h e 18S f o r m t o  t h e 14S s p e c i e s , and o f a s e c o n d 11S t e t r a m e r  9S s p e c i e s .  11S  appears t o i n v o l v e the  s e q u e n t i a l r e m o v a l o f one 11S t e t r a m e r give  with  of 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  a p p e a r a n c e o f an a d d i t i o n a l m o l e c u l a r AchE  (23,27,33,36).  toward  i s 1:1  11S enzyme ( 3 9 ) .  inhibitors  of the  no d i f f e r e n c e f r o m t h e 18S,  The 80,000 d a l t o n  subunit,  there-  as an i n d e p e n d e n t u n i t o f t h e enzyme  to the i n t e r a c t i o n with  ligands.  T h i s monomer i s  - 12  susceptible  -  to p r o t e o l y t i c cleavage  s i t e and a l l  proteases  except  generates  a 50,000 d a l t o n f r a g m e n t c o n t a i n i n g an a c t i v e  and  (40) p r o d u c e t h e 11S  form which  m i n o r f r a g m e n t s c o n t a i n i n g no a c t i v e s i t e s (41) .  teolysis the  collagenase  at a s p e c i f i c  11S  of c a t a l y t i c enzyme b u t  denatured Although  and  This  pro-  s u b u n i t s does not r e l e a s e fragments  from  i s detectable only after  subjected to d i s u l f i d e  the c o n v e r s i o n o f the  a g l o b u l a r 11S  form o c c u r s  the e x t e n t o f the c l e a v a g e of p u r i f i c a t i o n  and  18S  faster  D.  (12,40).  molecular  than p r o t e o l y t i c  forms  to  cleavage,  the c o n d i t i o n s  However, t h i s p r o t e o l y s i s s u b u n i t has  no  p r o p e r t i e s o f t h e enzyme  which  apparent  (38).  CATALYTIC MECHANISM Acetylcholinesterase  together inhibited  i s c l a s s i f i e d as a s e r i n e  w i t h o t h e r e s t e r a s e s and  hydrolase  p e p t i d a s e s , which are r e a d i l y  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 t h e s e r i n e r e s i d u e  active site. significant  The  amino a c i d  sequence about t h i s  r e s i d u e shows  s i m i l a r i t i e s t o t h e enzymes o f t h i s c l a s s  Several serine hydrolases their  14S  v a r i e s d e p e n d i n g on  storage.  the c a t a l y t i c  the enzyme i s  bond r e d u c t i o n  and  fragments a p o r t i o n of the c a t a l y t i c e f f e c t on  site  three-dimensional  crystallography  (43) .  (42).  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  s t r u c t u r e s as d e t e r m i n e d In the absence of X-ray  d e t e r m i n a t i o n of the t h r e e - d i m e n s i o n a l  by  X-ray  diffraction  s t r u c t u r e of AchE, i n -  f e r e n c e s a b o u t m e c h a n i s m so f a r have been b a s e d on  both  - 13  substrate  c a t a l y s i s and  r e s e r v a t i o n s , due i)  Substrate The  sists atic and  two  site  principal  (44) .  i n the  enzyme a t t h e various  The  of  t h e enzyme, subsites  anionic  site  active site  site  i s responsible  1-3  a f f e c t s the  site  (45-47).  This  i n the  simple  i s appropriate.  Most of  two  The  serine residue acetate  erate  the  limiting  binding  substrate  However f o r  scheme i s g i v e n  acetylcholine  (Ach)  the  in  reacts  forming a M i c h a e l i s o f Ach  i s accounted  site  (48).  but  With  the  the  leaving  the  site. to  rate  (49). is  The  regen-  i s the  s u c h as Ach hydrolysis  for  by  efficiently  deacetylation  f o r a good s u b s t r a t e ,  of  is  anionic  formed i s h y d r o l y z e d  concentrations,  show  implies that there  of AchE, d i s p l a c i n g c h o l i n e  This  is  of  undergoes n u c l e o p h i l i c a t t a c k  active surface. step  site  r a t e of h y d r o l y s i s  m o i e t y c o v a l e n t l y bound t o t h e e s t e r a t i c  a c y l enzyme s o  binding  Recent s t u d i e s  mechanistic  by C o u l o m b i c i n t e r a c t i o n s a t t h e o f i m i d a z o l e , Ach  ester-  p o c k e t m o d e l shown i n  Initially,  the  an  for  active region.  t h e enzyme a t i t s a c t i v e s u r f a c e  Menten complex.  and  or c h e m i c a l m o d i f i c a t i o n  differently.  binding  same f i g u r e  with  high  anionic  process.  inhibitors  p r e s e n t p u r p o s e , the  Figure  kinetics.  a c e t y l c h o l i n e s t e r a s e , con-  - an  actual catalytic  substrates  more t h a n one  aid  i n the  with  Reaction  t h a t the presence of  the  drawn from c h y m o t r y p s i n  o r i e n t i n g ammonium i o n , w h e r a s t h e e s t e r a t i c  involved  the  analogies  to important d i f f e r e n c e s  active site  of  -  At  -  Acetylcholine  14  -  hydrolysis  E + S ^ E - S — - > EA+ P — > E + P I  anionic  site  2.  esteratic  site  seryl  Figure  1-3:  The r e a c t i o n squeme o f acetylcholinesterase. E,enzyme; S , s u b s t r a t e ; Pt . c h o l i n e ; P 2 » a c e t i c  the  hydrolysis  of  acetylcholine  E S,Michaelis-Menten  acid;  EA,acetyl  enzyme.  complex;  by  - 15 -  i n h i b i t e d by the binding of a second s u b s t r a t e molecule to the a c y l enzyme EA  (50) .  The mechanism of t h i s i n h i b i t i o n  a complex EAS has wide a p p l i c a t i o n s .  forming  The a c t i o n of r e v e r s i b l e  i n h i b i t o r s and s u b s t r a t e r e a c t i o n s i s g e n e r a l l y e x p l a i n e d i n terms of b i n d i n g t o the a c y l a t e d as w e l l as t o the f r e e enzyme. ii)  I n h i b i t o r s and T h e i r I n t e r a c t i o n with  the Enzyme  A c e t y l c h o l i n e s t e r a s e i s now considered a d d i t i o n t o the a n i o n i c s i t e  t o possess, i n  i n the a c t i v e c e n t e r , a p e r i p h e r a l  a n i o n i c s i t e where l i g a n d s bind and e x e r t a r e g u l a t o r y i n f l u e n c e on the enzyme a c t i v i t y .  S i g n i f i c a n t evidence  of t h i s s i t e has been obtained  f o r the e x i s t e n c e  from k i n e t i c s t u d i e s using  n a t u r a l and s y n t h e t i c s u b s t r a t e s with v a r i o u s i n h i b i t o r s (21, 51,52), and more r e c e n t l y through n u c l e a r magnetic resonance (53) and f l u o r e s c e n c e  s p e c t r o s c o p i c measurements of l i g a n d  a s s o c i a t i o n with the enzyme having  (39,54).  Bisquaternary  two c a t i o n i c groups are c o n s i d e r e d  ligands  to span two a n i o n i c  s i t e s and the d i s t a n c e between two s i t e s can be estimated a t o 14 A, a d i s t a n c e of 10 carbon atoms apart decamethonium i o n , with  two  quaternary  (52,54,55).  The  ammonium groups sepa-  r a t e d by a 10 carbon a l k y l chain has thus a high a f f i n i t y t o wards AchE, and i s used f o r e l u t i n g AchE during by r e p l a c i n g the N-methylacridinium enzyme.  The binding modes of these  s c h e m a t i c a l l y i n F i g u r e 1-4  purification,  i o n i n i t i a l l y bound t o the l i g a n d s to AchE i s shown  (27,55).  - 16 -  active  center  peripheral esteratic  site  anionic  anionic  site  site  O  ft  CH.—C—0—CH9—CH9—N(CH,), 3 II c c. | 5 i + N(CHJ 3'3  acetylcholine  + N(CH3)3  (CH2),0  decamethonium N-methvlacridinium  Figure  1-4:  Binding  sites  ing s i t e s as w e l l  of acetylcholinesterase.  o f A c h E a r e shown w i t h  as l i g a n d s  orientation ever,  1  as a  used f o r AchE p u r i f i c a t i o n .  The  among t h e s i t e s  (39).  sites,  binding  and t h e h y d r o p h o b i c  D e c a m e t h o n i u m i s shown  d i s p l a c i n g the ligands  The d i s t a n c e  optimal  quinolinium. binds  sites  i n the series of bisquaternary  Cram claims t h a t  the peripheral  site  (42).  and  bind-  substrate, angular  region,  to bind  occupying  b e t w e e n two a n i o n i c  polymethylene-bis-trimethylammonium x  recognized  acetylcholine  i s n o t known.  anionic  Well  how-  t o both  either  site  i s deduced ligands  from  such as  polyethylene-bis-  the N-methylacridinium  ion also  - 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 d i v i d e d i n t o two groups depending on t h e i r mode o f a c t i o n .  The f i r s t group c o n s i s t s  of r e v e r s i b l e i n h i b i t o r s , and which r e a c t with AchE to form an e q u i l i b r i u m complex according  to the f o l l o w i n g scheme.  E + I g4V>EI Almost a l l r e v e r s i b l e i n h i b i t o r s c o n t a i n  a t l e a s t one p o s i t i v e l y  charged n i t r o g e n group, which binds t o the n e g a t i v e l y a n i o n i c s i t e on AchE i n h i b i t i n g  its activity.  charged  They vary i n  s i z e from simple i n h i b i t o r s such as d i v a l e n t c a t i o n s and t e t r a alkylammonium compounds, to l a r g e nueromuscular b l o c k i n g such as  d-tubocurarine and gallamine  i n h i b i t o r s should  (see F i g u r e  1-5). These  be u s e f u l t o o l s i n i n v e s t i g a t i n g the func-  t i o n a l p r o p e r t i e s o f the p e r i p h e r a l s i t e once t h e i r sites  drugs  binding  ( e i t h e r o f the two a n i o n i c s i t e s ) are p o s i t i v e l y i d e n t i -  fied.  T h i s 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  The  triethylammonium and propidium i o n s , and d i v a l e n t  c a t i o n s bind  s i t e s are i d e n t i f i e d with some c e r t a i n t 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  the 3-hydroxyphenyltrimethyl ammonium  inorganic  (54-57), whereas  (HPTA) and edrophonium ions  (see Figure 1-5) bind t o the a c t i v e a n i o n i c s i t e (51,54 ,58). However, r e p o r t s on i n h i b i t o r s such as N-methylacridinium, T e t r a methyl ammonium  (TeMA), gallamine and d-tubocurarine d i f f e r as  to which a n i o n i c s i t e s are i n v o l v e d 59-61).  (perhaps both)  (51,54,56,  Even among the l i g a n d s which seem t o occupy the same  s i t e s , a simple competitive  r e l a t i o n s h i p f o r occupation of a  - 18 -  0-(CH )—N-(C H )  3  0—(CH )—N—(C H )  3  2  2  NCH )2—N—(C2H5)3 2  N-methylacrtdintum  g a l 1 amine  (CH ) 3  d-tubocurarine  Figure  1-5:  Structure  of  AchE  inhibitors  2  2  2  5  5  - 19 -  s i n g l e s i t e does not always e x i s t Abou-Donia gallamine.  (54).  Roufogalis  (55)  and  (56) proposed a second p e r i p h e r a l binding s i t e for This confusion  r e s u l t e d from most s t u d i e s  r e s t r i c t e d to i n f e r e n c e s based on s t e a d y - s t a t e  being  k i n e t i c s of  h y d r o l y s i s of s u b s t r a t e s i n the presence of i n h i b i t o r s . k i n e t i c s behavior  of AchE and  the b i n d i n g of i n h i b i t o r s  a l s o a f f e c t e d by the i o n i c s t r e n g t h the source  of AchE (65)  and  aging  (39 ,54 ,62-64) , pH  (46).  The  of two  The are  (51),  sensitivity  AchE to i o n i c s t r e n g t h l e d Mooser to proposed the  the  of  existence  c o n f o r m a t i o n a l l y 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 The  later  supported  by Taylor  (66).  second group i s comprised of i r r e v e r s i b l e  which are e s t e r s or a c i d h a l i d e s of phosphoric,  inhibitors  carbamic  and  sulfonic acids.  They r e a c t by a scheme analogous to s u b s t r a t e  hydrolysis.  a c i d moiety of the  The  the e s t e r a t i c s u b s i t e forming  inhibitor  an acyl-enzyme intermediate  u n l i k e the acetyl-enzyme formed with s t a b l e to h y d r o l y s i s .  The  of minutes, f o r  of hours, and  f o r the  i n h i b i t o r y power of these  has  found a popular  and  as  bamate  the  phosphoryl-  (67) .  The  acid-transferring inhibitors  application in a g r i c u l t u r a l  nerve gases,. . Parathion  of  methanesulfonyl-  enzymes at r a t e s t h a t are too slow to be measured strong  which,  substrates, i s r e l a t i v e l y  h a l f - l i v e s f o r the h y d r o l y s e s  carbamyl-enzymes are of the order enzymes of the order  i s t r a n s f e r r e d to  and  insecticides,  1-naphthyl-N-methyl c a r -  ( c a r b a r y l ) , are used as i n s e c t i c i d e s .  Tabum and  Sarin,  - 20  causing respiratory the  p a r a l y s i s , were u s e d as war  S e c o n d W o r l d War.  nucleophilic Reversible  -  has  l o n g been o f  i n h i b i t o r s have been r e c e n t l y  spontaneous r e a c t i v a t i o n  e r a t e d by  (47,68).  of  but  by  research  t o r s , are  now  inhibitors  (TeMA, TeEA, and  others  acetylcholine  c o n s i d e r e d by  (HPTA and  hydrolysis  several  m a t i o n a l c h a n g e s i n d u c e d by  the  (47,  methanesulfonyl fluoride i s accel-  d e c e l e r a t e d by  s e e n on  interest.  p h o s p h o r y l AchE  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  effects  a  found to a c c e l e r a t e  c a r b a m y l and  Sulfonylation  some c a t i o n i c  piridinium),  during  Hence t h e d e v e l o p m e n t o f an a n t i d o t e ,  reactivator,  50,51,62,64).  gases  the  by  N-methyl-  gallamine) synergistic  pairs  of  inhibi-  authors to r e f l e c t confor-  b i n d i n g of c a t i o n i c  inhibitors  (21,47,51,58 ,69,70) . E.  SPECTROSCOPIC TECHNIQUES IN MECHANISM OF  their  the  by  an  the  acylation  or  acid-transferring  the  CATIONIC  group.  preference of  of  inhibitors  been d e r i v e d f r o m  enzyme a c t i v i t y ,  deacylation  i n t h i s k i n e t i c a p p r o a c h , as  the  or  active  their site  However, t h e r e are  the  h i n d e r e d by  the  ligands  a c h a r a c t e r i s t i c two  and  THE  b i n d i n g of c a t i o n i c  c a t a l y t i c m e c h a n i s m , has  i n h i b i t o r y e f f e c t on  f l u e n c e on  STUDY OF  ACETYLCHOLINESTERASE  M o s t i n f o r m a t i o n on h e n c e on  THE  two  interpretation binding  of  s i t e s for  and  either inserine  ambiguities results  is  cationic  s t e p c a t a l y t i c mechanism.  - 21  Spectroscopic the  interaction  1) n u c l e a r and  3)  provide  a useful tool  between i n h i b i t o r s  m a g n e t i c r e s o n a n c e , 2)  and  E  fluorescence  been u s e d f o r A c h E o n l y by K a t o  First,  the  Two  types  (NMR)  technique  (71-74).  This  l i n e w i d t h s of  inhibitor  protons  r e f l e c t the  changes i n c h e m i c a l  i n f o r m a t i o n on  s h i f t provide  e n v i r o n m e n t when b o u n d .  signal  The  o f bound i n h i b i t o r s  Hence, the  can  a l s o be not  small population  compared w i t h  in detail  binding.  He  reported  l i n e w i d t h s f o r bound i n h i b i t o r s . a t r o p i n e C-methyl proton  i s reported  5,600 Hz  (74).  Chapter  (72) IV  and  984  Hz  magnetic  de-  relation-  o f pH  and  ionic  different l i n e w i d t h of (71),  shown i n  t h a t t h e maximum p o s s i b l e l i n e w d i t h o f  and  and r e v e r s i b l e  21,000 Hz  a l s o be  inhibitors.  Atropine  example, the  It will  resonance  only  This  however t h r e e  t o be  binding  directly  be  irreversible  For  degree  The  free  II.  the e f f e c t  s t r e n g t h , as w e l l as t h e p r e s e n c e o f on  system.  i n Chapter  e s e r i n e were u s e d by K a t o t o s t u d y  inhibitors  the  seen  i n f o r m a t i o n p r e v i o u s l y m e n t i o n e d may  be d i s c u s s e d  with  Second,  obtained.  however be  duced under c e r t a i n c o n d i t i o n s of the ship w i l l  parts.  degree of s a t u r a t i o n of  r a t e s o f e x c h a n g e may  because of t h e i r  method  o f c h a n g e s a r e commonly o b s e r v e d .  specific  and  to date  interacting  of r e s t r i c t i o n of motion of t h e i r  sites  being:  spectroscopy,  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 t h e enzyme.  studying  resonance.  nuclear magnetic resonance  1  for  AchE, these  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  The has  techniques  -  bound  - 22 -  inhibitor ing  f o r 11S  results will  r e s u l t s obtained  AchE i s 400 Hz. be d i s c u s s e d  studying  i n C h a p t e r V, t o g e t h e r  i n the present  Fluorescence  The r e a s o n f o r K a t o ' s  spectroscopy  complex f o r m a t i o n  h a s been g a i n i n g p o p u l a r i t y i n The m o n i t o r i n g  by f l u o r e s c e n c e  which e i t h e r e x h i b i t g r e a t l y diminished a s s o c i a t i o n w i t h t h e enzyme  w i t h the  work.  l i g a n d a s s o c i a t i o n w i t h AchE.  ligand-AchE  conflict-  of  requires  quantum y i e l d s  ( 3 9 , 5 4 , 7 5 ) , o r whose  ligands upon  absorption  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 c e n c e upon b i n d i n g t o t h e enzyme  (66,76).  The b i n d i n g  s i t e s of  s e v e r a l f l u o r e s c e n t p r o b e s have been w e l l e s t a b l i s h e d . p h o n i u m and N - m e t h y l a c r i d i n i u m binding t o the a c t i v e center ionic site  strength binds (54).  show a marked p r e f e r e n c e f o r ( 3 9 ) , whereas p r o p i d i u m  a t low  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  T h e s e l i g a n d s a r e now  t i o n t o study  Edro-  used i n f l u o r e s c e n c e  titra-  t h e 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  non-fluorescent  quaternary  technique,  as o t h e r  convenient  measurement  ammonium l i g a n d s .  The  s p e c t r o s c o p i c methods, p r o v i d e s  fluorescence a more  of k i n e t i c parameters, since the l i g a n d  o f i n t e r e s t c a n be d i r e c t l y o b s e r v e d . enables a d i r e c t monitoring  For example, t h i s  method  o f t h e i n f l u e n c e o f t h e l i g a n d on  t h e m o d i f i c a t i o n o f t h e 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 obtained  activity  following extensive d i l u t i o n  (76).  In the study  by m e a s u r i n g r e s i d u a l enzyme of the r e a c t a n t  o f the a c y l a t i o n o f AchE, t h e r e  molecule  a r e two  - 23 -  k i n e t i c parameters which are often d i f f i c u l t T h e s e a r e 1) d i s s o c i a t i o n c o n s t a n t p l e x , and 2) t h e s p e c i f i c  by u s i n g  a substrate with  separate.  f o r t h e e s t e r - e n z y m e com-  rate constant  acyl-enzyme f o r the complex.  to  f o r the formation  T h i s c a n be a c h i e v e d  more  a l s o be o b t a i n e d  fluorescence  ( 6 6 ) , o r by t e m p e r a t u r e - j u m p r e l a x a t i o n k i n e t i c s  Although there for  limited and  (77) .  a r e a number o f s p i n - l a b e l l i n g  s u l f o n y l a t i n g , a c y l a t i n g and  ( 7 8 ) / none o f them to date.  ( w i t h one e x c e p t i o n )  has  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  free r a d i c a l being acetylcholine  phosphorylating been u s e d w i t h  a n i t r o x i d e group,  this  Spin-labelled  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 A b o u and t h e i r  k i n e t i c parameters of h y d r o l y s i s  ESR s t u d i e s on t h e s e i n d i c a t e d t h a t t h e a c t i v e  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 elsewhere  ( 7 8 ) . Wee  and S i n h a  site,  become i m m o b i l i z e d t o AchE.  tended conformation  sur-  and h a s been  observed that  n i t r o x i d e groups of s p i n - l a b e l l e d bisquaternary  binding  AchE  t o AchE h a s been  s t a b l e i n aqueous s o l u t i o n .  Donia e t a l (55,56),  reported  reagents  t o s p i n - l a b e l l e d analogs o f the r e v e r s i b l e i n h i b i t o r s  a c e t y l c h o l i n e , a l l of which contain  studied.  t o AchE  e i t h e r by . s t o p p e d - f l o w m e a s u r e m e n t s o f  of f l u o r e s c e n t l i g a n d s  available  simply  a f l u o r e s c e n t l e a v i n g group (75).  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 may  of  both  ammonium  ligands  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 This provided  evidence of binding  upon  i n an e x -  v i a a 2 p o i n t attachment i n v o l v i n g both  - 24  quaternary study  nitrogens  (79).  -  The method of s p i n - l a b e l l i n g  s u b s t r a t e and/or i n h i b i t o r  i n t e r a c t i o n s with the enzyme,  p r o v i d e s the same type of i n f o r m a t i o n as NMR,  the  spectrum  being a f f e c t e d by r o t a t i o n a l motional  c o n s t r a i n t s and  n e t i c and  advantage of ESR  e l e c t r i c environments.  ever, as compared with NMR,  One  the maghow-  i s a greater s e n s i t i v i t y .  It i s  thus p o s s i b l e to c a l c u l a t e the d i s t a n c e between the two on the enzyme u t i l i z i n g one  of the f o l l o w i n g :  l a b e l s , 2) a s p i n l a b e l with a diamagnetic s p i n l a b e l with a paramagnetic i o n . ESR  The  1) two  b i t o r , which enables  sites  spin  nucleus, or 3) a  high s e n s i t i v i t y of  a l s o means t h a t r e l a t i v e l y s m a l l c o n c e n t r a t i o n s of  tor can be used, r e s u l t i n g  to  inhibi-  i n a higher f r a c t i o n of bound  a d i r e c t o b s e r v a t i o n of the bound  inhi-  inhibitor.  Bulky s p i n l a b e l s however, can produce c o n s i d e r a b l e s t e r i c e l e c t r o n i c p e r t u r b a t i o n s of the parent molecule. necessary  I t i s thus  to demonstrate t h a t the s p i n l a b e l s bind to enzyme i n  the same f a s h i o n as a p a r t i c u l a r n a t u r a l i n h i b i t o r strate) .  and  (or a sub-  - 25 -  CHAPTER II THEORETICAL BACKGROUND  I n t e r p r e t a t i o n of t h i s t h e s i s i s based on equations developed from Bloch equations f o r the case where the popul a t i o n of one site.  site  i s very much g r e a t e r than that of the other  The r e l i a b i l i t y  of the r e s u l t  i s d i s c u s s e d i n terms of  the expected reasonable range of the t r a n s v e r s e r e l a x a t i o n time f o r a methyl group on a macromolecule. t h e o r e t i c a l background  The a p p r o p r i a t e  f o r the work i s thus e x p l a i n e d i n t h i s  chapter. A.  THE  LINESHAPE ANALYSIS FOR  EXCHANGE WHEN THE  A MOLECULE UNDERGOING CHEMICAL  POPULATION OF THE  TWO  SITES ARE  GREATLY  DIFFERENT As mentioned  i n the i n t r o d u c t i o n , the changes i n  NMR  chemical s h i f t s and r e l a x a t i o n times can be used to o b t a i n i n f o r m a t i o n on molecular i n t e r a c t i o n s i n s o l u t i o n .  NMR  spec-  troscopy p r o v i d e s a powerful t o o l f o r the study of the  inter-  a c t i o n of i n h i b i t o r s with enzymes. inhibitors  The c o n c e n t r a t i o n of bound  (those i n t e r a c t i n g with the enzyme), i s o f t e n  however, due  to the c o n c e n t r a t i o n of the enzyme.  o b s e r v a t i o n of NMR i n h i b i t o r s may  spectra i s therefore d i f f i c u l t .  limited  Subsequent Furthermore  undergo chemical exchange at a r a t e of the same  order of magnitude as the NMR  r e l a x a t i o n r a t e s ; t h i s slow  time  - 26  -  s c a l e phenomenon b e i 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 s p e c t r o s c o p i c methods. t o r s cannot t i o n can tra  Although  u s u a l l y be o b s e r v e d  t h e s p e c t r a o f bound  inhibitors.  t h e l i n e w i d t h o f t h e NMR  Bloch equations w i l l  be d e v e l o p e d ,  G u t o w s k y and tions with  certain McCall  to q u a n t i t a t i v e l y  later  and  i n f o r m a t i o n can  be  developed derived  t h a t t h e same  t h e s i s are  Consider  Connick  a system  M  z  in  A and  B.  (82)  X b a c k and  Using  reactions.  relationship equations,  (81).  s p i n systems,  f o r a three spin  where a r a p i d  cess t r a n s f e r s a nucleus environments  de-  lineshapes  The  interpreted using  i n t h e same manner f o r two  by S w i f t and  be  obtained.  r e v e r s i b l e , c h e m i c a l exchange  shown by M c C o n n e l l ,  r e s u l t s of t h i s  of  (80) u s e d t h e c l a s s i c a l B l o c h e q u a -  i n c l u d e the e f f e c t s of c h e m i c a l exchange  mental  spec-  classical  conditions w i l l  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 to  informa-  t h e NMR  peak,  r e l a t e n u c l e a r resonance  the r a t e s o f f a s t ,  I t was  inhibi-  In o r d e r t o e x p l a i n the e f f e c t  c h e m i c a l e x c h a n g e on  f i n e d under which  other  directly, quantitative  be d e d u c e d u n d e r c e r t a i n c o n d i t i o n s f r o m  of free  from  experi-'  equations as  those  system.  r e v e r s i b l e molecular f o r t h b e t w e e n two  pro-  molecular  the B l o c k ' s n o t a t i o n , l e t u, v  denofae t h e c o m p o n e n t s o f t h e n u c l e a r m a g n e t i z a t i o n w h i c h phase w i t h  the component o f the r f f i e l d  f r e q u e n c y to, out o f phase w i t h t h i s  rptating  rotating  the d i r e c t i o n of the l a r g e s t a t i o n a r y f i e l d , L e t M^  be  t h e v a l u e o f M^  at equilibrium.  at  rf field,  are  angular  and  respectively.  Then t h e  and  Block  along  - 27  equations i n the  frame r o t a t i n g at frequency to, are  M •  -  dM  - &  (M  "  TTF  " %  H  1  -  -  V  ( l b )  - M )  < '  T,  lc  where w  i s the  nuclear resonance frequency  which  and  are the l o n g i t u d i n a l and t r a n s v e r s e  T  2  times r e s p e c t i v e l y .  By  introducing  the  (io = Y N 0  H  D  ) »  i n  relaxation  notation  M = u + iv (la) and  + {(  §f where M  (lb) reduce  z  Now  -  W o  = M  o  W  to  ) i - ± } M  = i  f o r a weak f i e l d  W l  M  (1)  0  H,  1 at steady s t a t e , J  c o n s i d e r a chemical exchange.  I f the p r o b a b i l i t y  the nucleus jumping  from s i t e A to B i s P g 6 t ,  P  are  B A  6t,  and  M  A  respectively, be w r i t t e n  and  Mg  A  the  dM,  St  a  -=  _P  " AB  M  + P  A  BA  M  B  dM„ E = -P M + P M dt BA B AB A +  versa  components of M at s i t e s A and  then the change i n M  as  or v i c e  A  and  M  g  due  of  to jumping  B can  - 28 -  Equation  (2) c a n be w r i t t e n  c o n t r i b u t i o n from  f o r M^ and M  chemical  exchange  dM  „  St  +  +  dM  AB "  P  <V A  i A  M  " BA B P  <T^  +  where A w  =  l oA  < >  ^l oB  < >  i w  M  31  +  P  BA "  1 A  ^B) B M  - AB A P  M  =  M  3b  = O J A - OJ a n d AWg = co - to.  A  B  At the steady  s t a t e , f^A = ^ B = 0 i n e q . (3a) a n d  the simultaneous  equations  T T 7( T TB"A A -B "V A B B A< < " +  P  P  +  i A  P  (3b).  dt  dt Solving  M  „  1  ~dF  P  t o include the  g  M  i A  o VA  (  forM  A  then  T-T B7 A oBBA> ' l P  +  MP  1 W  yields  V  i A  M = B  However s i n c e A i s o v e r w h e l m i n g l y M  oB*  H  total  e  n  c  e  P  BA OB M  C  A  N  B  E  i9  n o r e c  d o m i n a n t t o B, t h e n M  3 compared w i t h  P  M B A  0  ^*  T  n  Q A  >>  e  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 c a n be wr i t t e n a s (  P  T7~  + P  J  BA - <V oA i A  M  ;  AB BA P  +  P  AB "  l A w  B  }  j +  ; P  BA "  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 , t h e o u t o f p h a s e component o f M, t h e i m a g i n a r y  terms a r e c l e a r e d from  From t h e r e s u l t i n g and  rearranged  the denominator.  equation, the imaginary  to give  terms a r e s e l e c t e d  - 29 -  C-  M  l )" oA  v= 2  < T T - BA> ( f +  P  2+  Aw  B  The only assumption over B.  made u n t i l now i s t h a t A i s i n l a r g e excess  The numerator and the term  are e s s e n t i a l l y  frequency  i n the f i r s t  independent.  v w i l l thus occur when the second  term  bracket  The maximum value of i n the denominator i s  zero, and the h a l f - w i d t h at h a l f - h e i g h t f o r t h i s L o r e n t z i a n like in  l i n e s h a p e i s given t o a good approximation,  the f i r s t  bracket i n the denominator.  by the term  The experimental  h a l f - w i d t h at h a l f - h e i g h t i s given by the symbol  1.  (4)  Under l i m i t i n g c o n d i t i o n s , s i m p l i f i c a t i o n ever, leads t o more u s e f u l e q u a t i o n s .  of equation 4 how-  These are shown i n  Table I I - l together with the c o n d i t i o n s of l i m i t i n g cases and the i n f o r m a t i o n o b t a i n a b l e . In the t a b l e A t o  B  = to - c o i s r e p l a c e d by Ato = t o - t o • B  A  B  In the slow exchange r e g i o n , c u , can be r e p l a c e d by to, s i n c e  -  Table  30  11-1  Expected r e l a x a t i o n times when A i s t h e d o m i n e n t  speed of exchange  -  i n presence of  speceis in  the chemical  solution  information  observation  condition  exchange  obtainable  (})*  L» » %  ,  1 = T1 T  P^A  2  +  2A  f  f*p BPBA P  slow < T > B »  * -  A W  P  =1  BA  +  BA  ,  A  0J  P  AB  P (4-a)  T  2  fast  P  BA  W  T  »  1 P  2B  BA  T  _ 2  1 T  B ^  f  +  2A  P  BA f4-b)  very fast  T1 " 2B  p  D  BA  >  >  (  T1  )  2  2B  '  A  U  F  L  1=1 T  2  T  + f l  2A  B T  2B  1 T  '  Pr  ,  '2B  K DU  C4-c) slow  T  T' l B  »  TI B  «  P  Bb AA  1=1 T  l  T  +  f  P  BA  1A  P  AB  l fast  1  P  BB AM  1=1 T  l  T  1A  +  f l BT  1  1B  T1  1B  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 and e x p r e s s e d P  P  AB  AB +  P  BA  '  as  K  DU  -  only  a s i n g l e l a r g e peak due  condition  QT]  >>  QBJ  , and  -  31  t o A would resonance  a l m o s t e q u a l t o t h a t o f p u r e A. exchange r e g i o n be c l o s e  since  frequency  as P  would  (~l  2  - T^T —) i s g o v e r n e d 1  rate  frequency, then  the  by t h e r a t e o f c h e m i c a l  2A  In the f a s t r e g i o n ,  by b o t h t h e o f f - r a t e and  the l i n e broadening  i s controlled  the change i n the p r e c e s s i o n a l  fre-  q u e n c y , w h e r e a s i n t h e v e r y f a s t r e g i o n , c o n t r o l i s by t h e relaxation process The  e q u a t i o n s shown i n T a b l e  a s i m i l a r manner as T , 2  species  2 B  I I - l f o r the l o n g i t u d i n a l r e -  peak a r e o b t a i n e d f r o m e q u a t i o n l c where t h e s o l e a s s u m p t i o n  i s that  A i s much more c o n c e n t r a t e d t h a n B i n t h e s o l u t i o n .  As e x p e c t e d of  T  only.  l a x a t i o n time of observed in  A.  change  f r o m B t o A ) * i s much s l o w e r t h a n e i t h e r t h e  broadening  exchange.  peak  t o l a r g e excess of pure  of 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 line  fast  - the r a t e molecules  g A  be  i n the  the frequency of the averaged  When t h e o f f - r a t e ( d e f i n e d  the  (to) w i l l  This i s also true  t o t h a t o f p u r e A a l s o due  environment  be o b s e r v e d w i t h  f r o m e q u a t i o n l c , e q u a t i o n s f o r T^  t h e c h e m i c a l s h i f t d i f f e r e n c e i n t h e two  e q u a t i o n s f o r T^  are  sites.  independent However  a t b o t h e x t r e m e l i m i t s o f e x c h a n g e show t h e  same f o r m s as o n e s 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 u s e d s i n c e e q u a t i o n s d e 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 t h e i n t e r a c t i o n b e t w e e n i n h i b i t o r s and e n z y m e s . In t h i s s i t u a t i o n B A d e s c r i b e s t h e 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 t h e enzyme. P  - 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  with AchE i s that  f o r choosing i n h i b i t o r s  interacting  there should be present at l e a s t one methyl  group t o y i e l d a 3 - f o l d  increased s i g n a l t o 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 s p e c t r a  with no s p l i t t i n g s from s c a l a r  T h i s g i v e s another advantage s i n c e laxation  times T-^ and T  2  the expected magnetic r e -  f o r a methyl group r i g i d l y  to a s p h e r i c a l macromolecule were c a l c u l a t e d Marshall spin  (83,84).  of T  goes t o z e r o .  i  T l  T  c  2  The r e l a -  with the r o t a t i o n a l c o r r e l a t i o n time i s p l o t t e d  2  i n F i g u r e I I - l , according t o the f o l l o w i n g from t h e i r  for a three-  the e f f e c t of c r o s s - c o r r e l a t i o n , and  took the l i m i t that c r o s s - c o r r e l a t i o n tionship  attached  by Werbelow and  They considered the r e l a x a t i o n  system, i n c l u d i n g  coupling.  equation obtained  calculation.  -3  4 2  5T  V  6 10r  b  1  T  c c  1 +  2T  2 W  2 T  C  i s the r o t a t i o n a l c o r r e l a t i o n  2 2' 1 + 4co T  (  '  C  time i n terms of the r o t a t i o n a l  d i f f u s i o n constant f o r a sphere and r i s the proton-proton d i s t a n c e i n a methyl group.  The r o t a t i o n a l c o r r e l a t i o n  i n the magnetic resonance i s expressed, f o r a s p h e r i c a l c u l e , as  time mole-  -  33  -  Log[l/T2]  10 Log[-cc0  Figure  11-1:  A plot  of  A curve 1/T2  for  1/T2  vs.  reciprocal rotational  i s obtained a methyl  from Equation  group  time.  (5), which c a l c u l a t e s  r i g i d l y attached  m o l e c u l e whose r o t a t i o n a l  correlation  to  c o r r e l a t i o n time  a spherical is  ~cc.  -  34  -  where R, n, k and T are the r a d i u s of the sphere, the s o l u t i o n v i s c o s i t y , the Boltzmann constant (85).  Hence i f the molecular  and the absolute  weight, the p a r t i a l  temperature specific  volume of the molecule, and the s o l u t i o n 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 c a l c u l a t e d , and thus the expected values C.  for T . 2  PLOTS TO OBTAIN BOUND LINEWIDTH There are two methods to deduce the bound NMR  of an i n h i b i t o r  from the l i n e w i d t h a c t u a l l y observed when the  rate of exchange i s i n the very it  linewidth  fast region.  In both methods,  i s assumed t h a t the c o n c e n t r a t i o n of the i n h i b i t o r  greater  than the c o n c e n t r a t i o n of the enzyme.  the same equation  of T  f o r the very  2  i s much  S t a r t i n g from  f a s t exchange region i n  Table I I - l ; 1 T  2  -  1 T  U  +  2A  Gerig obtained  1  B T  the f o l l o w i n g e x p r e s s i o n ( 8 6 ) .  ± - -L. = T  2  T  2A  2B  I  E  o  +  %  In the above equation enzyme and i n h i b i t o r a t i o n constant EI.  T  E  Q  (  7)  2B  and I  Q  are the i n i t i a l c o n c e n t r a t i o n of the  r e s p e c t i v e l y , with K  representing  1 1 7 p — and 7 = — now  f r e e and bound  • _L_  °  being  the d i s s o c i -  for equilibrium E + I %  C represent  inhibitors.  D  E I  J  . the l i n e w i d t h s of n u c l e i i n  - 35 -  K a t o made a n o t h e r o f t h e bound inhibitor,  assumption;  inhibitor  namely t h a t the l i n e w i d t h  i s much g r e a t e r t h a n  and t h u s o b t a i n e d  t h a t of the f r e e  the f o l l o w i n g equation (73).  1 Top, =  E  V  o T  For  both  2  ~ D  <>  K  8  " ^2A  equations,  t h e bound l i n e w i d t h i s o b t a i n e d  the g r a d i e n t o f a s t r a i g h t  line.  Although  make two a s s u m p t i o n s t o o b t a i n e q u a t i o n a d v a n t a g e t h a t t h e known v a l u e o f t h e v a l u e o f —-. 2  Furthermore,  as  i t was n e c e s s a r y  7, t h i s e q u a t i o n  to  has an  i s not required t o o b t a i n  i t i s p o s s i b l e t o o b t a i n the K  i  value tion  D  as t h e y i n t e r c e p t o f a s t r a i g h t 7.  The K  D  value obtained  however, as g e n e r a l l y K  D  T h e s e two e q u a t i o n s r e s u l t s and c a l c u l a t e  << I  Q  l i n e obtained  from equa-  i n t h i s manner i s n o t a c c u r a t e .  a r e used i n Chapter  t h e bound  linewidth  T  ^ 2B  IV t o p l o t the  - 36  -  CHAPTER I I I EXPERIMENTAL METHODS  A.  P U R I F I C A T I O N OF  i)  E x t r a c t i o n o f AchE f r o m E l e c t r i c Live e l e c t r i c  ACETYLCHOLINESTERASE  e e l s were k i l l e d  Media  by p a c k i n g them i n t o a  i c e - w a t e r m i x t u r e f o r h hour  crushed  E e l i n High S a l t  and  t h e main  o r g a n s were removed by d i s s e c t i o n , y i e l d i n g  electric  400-800 g p e r e e l . 3  Fresh e l e c t r i c cubes. in  t i s s u e was  Electric  c u t up  into approximately  t i s s u e p i e c e s n o t used  l i q u i d n i t r o g e n and  stored  2-cm  i m m e d i a t e l y were  at dry i c e temperature  frozen  until  needed. The  following  autolysis  (27,40,87).  homogenized Blender  f o r 30 s e c o n d s  frigerated  f o r h hour  P i e c e s o f f r e s h or f r o z e n  a t low s e t t i n g . a t 10,000 rpm  c e n t r i f u g e and  the t o t a l a c t i v i t y  as b e f o r e .  Na EDTA  seconds  2  pH  were  i n a Waring  homogenate was  The  r e s i d u e was  o f 5% s u c r o s e s o l u t i o n  buffer  7.0  tissue  then  i n t h e S o r v a l RC2B r e -  poured o f f .  T h i s r e s i d u e was  volume o f h i g h s a l t  The  to avoid  supernatant l i q u i d containing  was  g e n i z e d w i t h two v o l u m e s  2 mM  c a r r i e d o u t a t 4°C  i n one v o l u m e o f 5% s u c r o s e s o l u t i o n  centrifuged  of  p r o c e d u r e was  then homogenized  (2 M N a C l , 20 mM  and  rehomo-  centrifuged  i n a 3-5 sodium  10-20%  fold  phosphate,  a d j u s t e d a t room t e m p e r a t u r e ) f o r 30  and c e n t r i f u g e d a t 31,000 rpm  f o r 6 h o u r s i n t h e 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 . n a t a n t was  r e t a i n e d and  w i t h the r e s i d u e . accounted  Supernatants  fer  (20 mM  filtered  sodium phosphate,  to centrifugation.  omitted.  i n the f i r s t  pH  7.0)  t o g i v e the f i n a l  ii)  extract  chromatography.  by l e t t i n g  t h e homo-  i n the  refrigerator  further extraction  d i d n o t a f f e c t t h e r e c o v e r y and  o f A c h E by a f f i n i t y  o f AchE by A f f i n i t y  the  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 S e p h a r o s e 2B  column  i n a f f i n i t y c h r o m a t o g r a p h y had  Webb a c c o r d i n g t o p u b l i s h e d m e t h o d s of s p e c i f i c ,  (18).  affinity  been p r e p a r e d The  When a c r u d e  r e v e r s i b l e AchE  inhibitors agent,  e x t r a c t o f AchE i s a p p l i e d  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 , AchE i s s e l e c t i v e l y  retained  and  by  procedure i n -  t o a S e p h a r o s e r e s i n m a t r i x by means o f t h e c o u p l i n g cyanogen bromide.  puri-  Chromatography  P r e p a r a t i o n of the  v o l v e s the attachment  alter  columns.  a)  m a t r i x used  re-  was  T h i s d e l a y i n t h e e x t r a c t i o n d i d n o t seem t o  Purification  buf-  I n t h i s c a s e , 90% o f t h e AchE was  e x t r a c t i o n and  t h e f o r m o f AchE and fication  The  t h e n d i l u t e d w i t h an e q u a l v o l u m e o f no s a l t  stand overnight i n high s a l t buffer  covered  original  through g l a s s wool.  Some o f t h e e x t r a c t s were p r e p a r e d  prior  which  i n the  o f 1 M N a C l , as r e q u i r e d f o r t h e e n s u i n g a f f i n i t y  genate  repeated  from both e x t r a c t i o n s ,  f o r 60-70% o f t h e t o t a l AchE a c t i v i t y  was  super-  t h e h i g h s a l t e x t r a c t i o n was  h o m o g e n a t e , were c o m b i n e d and filtrate  The  t h e enzyme may  t h e n be  to  the eluted  -  -  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 with a stronger the  liquid  attached  t h i s c a s e had  t o the m a t r i x .  ymol/mfi, p a c k e d g e l , w h i c h g i v e s  and  a good s p e c i f i c  posable  modified  mM  and  After HC1  packed  e a c h use  pH  7.0)  i n 10 ml  by  12 h o u r s .  f l u s h i n g the  washed w i t h  passed  dis-  (20  packed  After thoroughly  (approximately  One  a 5M  Guanidine-  column volume  t h r o u g h t h e c o l u m n and  stopped t o l e t the g e l stand  graphy b u f f e r  recovery  buffer.  t h e c o l u m n was  G u a n i d i n e - H C l s o l u t i o n was  minimum o f  ~  e q u i l i b r a t e d with chromatography b u f f e r  w i t h the  in  b o t t o m d i s c s made f r o m p o r o u s  s o l u t i o n i n chromatography b u f f e r .  f l o w was  of  t h e most e f f i c i e n t  S e p h a r o s e 2B g e l was  s o d i u m p h o s p h a t e , IM N a C l ,  column t h o r o u g h l y  used  (27).  syringes with f i t t e d  polypropylene  a f f i n i t y matrix  ligand concentration  0.4  The  than  The  a N-methylacridinium  activity  binding constant  i n the  the  solution for a  washing w i t h  five-fold  of  chromato-  volume) the column  was  ready for r e l o a d i n g . The repeated  p e r f o r m a n c e o f t h e c o l u m n d e t e r i o r a t e d however use,  due  t o b u i l d - u p o f m a t e r i a l on  growth of b a c t e r i a . column p a c k i n g T h i s was NaCl,  with  done by  5 mM  Therefore trypsin  i t was  after  top of the g e l  necessary  to t r e a t  a c e r t a i n number o f  suspending the g e l i n t r y p s i n b u f f e r  T r i s HC1,  pH  to a f i n a l concentration  7.5)  and  o f 0.04  adding freeze d r i e d mg/m£.  after  Then t h e  and  the  runs. (0.5  M  trypsin  solution  was  -  39  -  i n c u b a t e d a t room t e m p e r a t u r e f o r a few d a y s .  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 c h r o m a t o g r a p h y f o r e u s e and g a v e  j u s t a s good  b u f f e r be-  a p e r f o r m a n c e as n e w l y p r e p a r e d  gel. b)  Running o f t h e 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 a s 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 o n t o t h e c h r o m a t o g r a p h y c o l u m n s f l o w o f l e s s t h a n one c o l u m n volume  per hour.  After  at a  loading  was c o m p l e t e d , t h e n o n - a d s o r b e d 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 t h e same f l o w r a t e u n t i l  r e a d i n g was 0.3 o r l e s s  t h e &280  (approximately f i v e volumes).  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 c o l u m n volume Decamethonium Bromide  i n chromatography  buffer  tography buffer  o f 20 mM  at a flow  o f 0.1 c o l u m n v o l u m e s p e r h o u r and was c o l l e c t e d o f a p p r o x i m a t e l y 1 ml.  AchE  in fractions  The e l u t i o n was c o m p l e t e d w i t h  a t t h e same f l o w r a t e .  rate  chroma-  A l l o p e r a t i o n s were  done a t 5°C. The  a b s o r b a n c e a t 280 nm o f e a c h f r a c t i o n was d e t e r m i n e d  and t h e e l u t i o n p r o f i l e was p l o t t e d . file  i s given  measured cific  i n Figure I I I - l . )  The a c t i v i t y  a c t i v i t i e s were  a t 5°C t h r e e t i m e s a g a i n s t b u f f e r  each, i n order  and t h e s p e -  calculated.  f r a c t i o n s w i t h low s p e c i f i c  and d i a l i s e d  pro-  o f AchE was  f o r t h e f r a c t i o n s a r o u n d t h e m a j o r peak  a c t i v i t i e s were The  (A t y p i c a l e l u t i o n  t o remove t h e d e c a m e t h o n i u m  combined  f o r 24 h o u r s  bromide.  The  - 40 -  Figure  111-1:  An e l u t i o n  profile  of  AchE f r o m  an a f f i n i t y  column.  - 41 -  r e m a i n i n g s o l u t i o n was t h e n d i l u t e d t e n t i m e s and r e p u r i f i e d by  affinity  chromatography as d e s c r i b e d  A c h E s o l u t i o n was c o m b i n e d w i t h  above.  the f r a c t i o n s of high s p e c i f i c  a c t i v i t y of the o r i g i n a l p u r i f i c a t i o n , giving approximately protein iii)  The r e s u l t i n g  9 mM o f a c e t y l t h i o c h o l i n e  an a c t i v i t y o f  hydrolized  p e r mg o f  per min.  Acetylcholinesterase All  activities  Assay  f o r A c h E i n t h i s t h e s i s were a s s a y e d b y t h e  p h o t o m e t r i c method d e v e l o p e d by E l l m a n e t a l ( 8 8 ) , u s i n g t h e substrate  analogue a c e t y l t h i o c h o l i n e .  method i s t h e measurement o f t h e r a t e  The p r i n c i p l e o f t h e of production of t h i o -  choline  as a c e t y l t h i o c h o l i n e  plished  by o b s e r v i n g t h e e n s u i n g r e a c t i o n  with  1  the  i s hydrolyzed.  5,5 -dithiobis-2-nitrobenzoic yellow  colour  0.1  acid.  hydrolysis,  as t h e r e a c t i o n  with  produces  The r a t e o f of acetylthio-  DTNB i s s u f f i c i e n t l y hydrolysis.  a s s a y 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  M s o d i u m p h o s p h a t e pH 8.0, 25 u£ o f 0.075 M  line, and  (DTNB), w h i c h  i s a d i r e c t measure o f t h e r a t e  f a s t compared t o t h e a c e t y l t h i o c h o l i n e The  o f the t h i o l a t e anion  anion of 5-thio-2-nitro-benzoic  formation  choline  acid  T h i s i s accom-  acetylthiocho-  100 \il o f 0.01 M DTNB i n 0.1 M s o d i u m p h o s p h a t e pH 7.0  50 \il o f enzyme s o l u t i o n  i n 0.1 - 1 M N a C l c h r o m a t o g r a p h y  buffer  i n t o a 1 cm c u v e t t e and m i x i n g  crease  i n a b s o r b a n c e a t 412 nm was m e a s u r e d  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 .  i t thoroughly. i n a Zeiss  The i n PMQII  AchE s o l u t i o n s were d i l u t e d  - 42 -  to ensure t h a t there was enough s u b s t r a t e  to maintain a l i n e a r  r a t e o f h y d r o l y s i s f o r at l e a s t 2 minutes. Enzyme a c t i v i t i e s are reported  i n u n i t s , where each u n i t of en-  zyme w i l l change the absorbance a t 412 nm by 0 .1 ml cific  activity  min ^.  i s c a l c u l a t e d i n mmole o f a c e t y l c h o l i n e  l y z e d per mg of p r o t e i n per min, where the p r o t e i n tion  (mg/m£) f o r the AchE was determined using  1% value £280nm  =  -^.O  hydro-  concentra-  the published  (13) and the r a t e o f h y d r o l y s i s o f a c e t y l -  t h i o c h o l i n e was c a l c u l a t e d using  the e x t i n c t i o n c o e f f i c i e n t f o r 4  the t h i o l a t e anion, e^^nm B. i)  Spe-  =  1*36 x 10  CONVERSION OF AchE TO U S Trypsin Digestion  -1 M  -1 cm  a t pH 8.0.  FORM  AchE s o l u t i o n s obtained as above were t r e a t e d with t r y p s i n to form the g l o b u l a r against  l i s form.  The AchE s o l u t i o n was d i a l y s e d  100 volumes of t r y p s i n b u f f e r  Then t r y p s i n d i s s o l v e d  i n the same b u f f e r was added to the AchE  solution to a f i n a l concentration AchE s o l u t i o n  at 4°C twice f o r 24 h r s .  of 1 mg T r y p s i n  per 25 ml  approx. 0.7). The s o l u t i o n was incubated  at room temperature f o r 20 minutes.  The r e a c t i o n was terminated  by adding soy bean t r y p s i n i n h i b i t o r (STI), where STI had a concentration  of 2 mg per 25 ml of AchE s o l u t i o n .  (The a c t i v i t y  of AchE was measured before and a f t e r the d i g e s t i o n and i t was found that n e i t h e r  t r y p s i n d i g e s t i o n nor the presence of STI  changed the a c t i v i t y o f AchE).  - 43 -  The graphy.  s o l u t i o n was f u r t h e r p u r i f i e d by a f f i n i t y  chromato-  To t h i s end i t was d i l u t e d to 400 u n i t s of a c t i v i t y  per ml with 0.1 M NaCl chromatography b u f f e r and then  loaded  onto the e q u i l i b r a t e d column and washed and e l u t e d with 20 mM decamethonium bromide as d e s c r i b e d  i n S e c t i o n A - i i - b , however  0.1 NaCl chromatography b u f f e r was used i n s t e a d of r e g u l a r IM NaCl chromatography b u f f e r 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 f o r l a t e r batches, t h i s step was omitted s i n c e i t was found t h a t i n c u b a t i n g f o r a somewhat longer  o r i g i n a l AchE s o l u t i o n s with t r y p s i n  p e r i o d o f time  (approx. 1 hour) leads to  conversion  i n t o the 11S form i n almost 100% y i e l d .  conversion  or l o s s of a c t i v i t y could be  ii)  No f u r t h e r  detected.  C h a r a c t e r i z a t i o n o f Converted AchE For each p r e p a r a t i o n  extent  of conversion  of t r y p s i n t r e a t e d AchE s o l u t i o n , the  t o the 11S form o f AchE was t e s t e d by  i s o k i n e t i c sedimentation i n a sucrose g r a d i e n t  which i s a r -  ranged so that macromolecules of d i f f e r e n t molecular weight sediment a t constant nentional previously  rates, respectively.  The r e q u i r e d  expo-  sucrose g r a d i e n t , whose range had been c a l c u l a t e d (87), was prepared by pumping a 29.3% sucrose  s o l u t i o n i n chromatography b u f f e r  i n t o a 10% sucrose s o l u t i o n  i n the same b u f f e r , which i n turn was pumped at e x a c t l y the same speed  (use of a p e r i s t a l t i c pump) i n t o the g r a d i e n t  (7.3 cm c e l l u l o s e n i t r a t e tubes f o r Beckman SW41  swinging  tubes  - 44 -  bucket r o t o r ) . thin  Prepared gradients  were c o o l e d  t o 4°C and a  l a y e r o f A c h E s a m p l e ( a l s o p r e c o o l e d ) was  carefully  a p p l i e d onto the top of the sucrose g r a d i e n t . contained  100 u& o f AchE s o l u t i o n c o n t a i n i n g  of a c t i v i t y lase  and 100 y£ o f s t a n d a r d s ;  i n 10% s u c r o s e s o l u t i o n .  Each sample 300-1000  f3-galactosidase  The s e d i m e n t a t i o n  units and  cata-  constants  of  t h e s e p r o t e i n s a r e 15.9S and 1 1 . 3 S , r e s p e c t i v e l y ( 2 8 , 2 9 ) . gradients trifuge tion  were c e n t r i f u g e d  the g r a d i e n t s  emptying  collecting  AchE was d e t e c t e d  B-galactosidase  d a t a from other  i s converted  was d e t e c t e d  III-2.  reports  by a s p e c i f i c  buffer  as d e s c r i b e d  t r y p s i n , are p l o t t e d  the  assay taken against  I t i s c l e a r from the f i g u r e  The 11S AchE was  100% o f AchE concentrated  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  H o w e v e r , when t h e enzyme c o u l d  i m m e d i a t e l y , i t was s t o r e d  activity  w h i c h was  (27,40) t h a t n e a r l y  t o t h e 11S f o r m .  without delay.  (20 mM  slowly  by i t s a c t i v i t y , w h i l e  and a f t e r t h e t r e a t m e n t w i t h  2  inserting a  f r a c t i o n s of approximately  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 ,  into D 0  the c e n t r i f u g a -  l o c a t e d by t h e c h a r a c t e r i s t i c a b s o r b a n c e a t 405  f r a c t i o n number i n F i g u r e and  Following  were f r a c t i o n a t e d by c a r e f u l l y  i t s contents,  c a t a l a s e was  before  23-50 u l t r a c e n -  t u b e down o n t o t h e b o t t o m o f t h e t u b e and  0.5 ml v o l u m e .  (8).  i n a Beckman  f o r 19-20 h r s a t 40,000 rpm.  capillary  nm.  a t 4°C  The  (up t o one month)  s o d i u m p h o s p h a t e , pH 7 . 0 ) .  n o t be u s e d  i n 4 M NaCl  buffer  - 45 -  Figure  III-2:  Sucrose gradient after  (b)  p r o f i l e o f AchE b e f o r e  treatment  with  trypsin.  (a)  and  - 46  -  C.  PREPARATION OF  i)  C o n c e n t r a t i o n o f t h e Enzyme w i t h t h e A m i c o n 11S  AchE  A c h E SAMPLES IN D 0  (10 mil s o l u t i o n , see :  filter),  w h i c h had  with chromatography b u f f e r .  apparatus  The  s o l u t i o n was  filter  r e t a i n s macromolecules with a molecular  D0 2  buffer  (equipped  concentrated  n i l volume by a p p l y i n g a p r e s s u r e  was  o f 14-20  psi.  weight  t o p p e d up w i t h a p p r o x i m a t e l y  (0.1 M N a C l ,  20 mM  and  the s o l u t i o n  This process  was  repeated  added t o a f i n a l A g ^ 2  to  18  uM  account By  then  and  loss in specific recovered  buffer, while  activity.  buffer  2  This  This  was  corresponds t a k i n g into  recovered  t h e r e was  no  after  appreciable  i n d i c a t e s t h a t a l l t h e enzyme  i s c a t a l y t i c a l l y a c t i v e and  probably  has  not  changed  form. The  concentrated  in  a plastic  two  weeks.  vial  enzyme s a m p l e i n D 0  a t 4°C  and  used  7.0;  STI.  t h i s method 6 5 - 7 5 % o f t h e enzyme was 2  pH  n i l volume.  s u b u n i t s of AchE, a f t e r  the c o n t r i b u t i o n s from t r y p s i n  into D 0  D0  of  isotope ef-  to almost  r e a d i n g o f a r o u n d 3.5.  o f 80,000 c a t a l y t i c  concentration  its  o n c e more and  (UM10  10 ml 2  again concentrated  to  > 10,000.)  sodium p h o s p h a t e , i n D 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 fect)  filled  been e q u i l i b r a t e d p r e v i o u s l y  almost  Then t h e a p p a r a t u s  Filter  above s e c t i o n ) was  i n t o an A m i c o n D i a f l o U l t r a f i l t e r a t i o n w i t h an UM10  BUFFER  2  2  b u f f e r was  stored  f o r NMR  experiments  within  -  ii)  Other Methods f o r C o n c e n t r a t i n g The  f o r NMR  2  t h r o u g h s e p h a d e x G25  studies.  time.  However i t was  found  t h a t t h e enzyme was  solution  c o n d i t i o n s , when t h e at l i q u i d N  a c t i v i t y could  no  f r e e z i n g , and  be  recovered.  s a m p l e was  The  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 l o s s of p r o t e i n could  be  advantage of the  concentrated was  placed  injecting  sample of on  D0 2  t i o n was  layer  during  b u f f e r o n t o the  counted using  counter.  the  I t was  The  o f enzyme  process,  since  buffer  2  s p e c i f i c g r a v i t y o f D 0.  A  2  (H 0) 2  solution  b u f f e r by  bottom of the  carefully  tube c o n t a i n i n g the  same manner as d e s c r i b e d  of H 0 2  mixing  added t o t h e with D 0 2  r a d i o a c t i v i t y of t r i t i u m  a Nuclear  enzyme  enzyme i n t o D 0  A s m a l l amount o f t r i t i u m was extent  most  i n 5% g l y c e r o l  A f t e r c e n t r i f u g a t i o n a t 40,000 rpm  t o monitor the  stable  of o r i g i n a l  the  2  centrifugation. was  35%  enzyme i n a q u e o u s  f r a c t i o n a t e d i n the  tion B - i i .  frozen  t o p o f a l a r g e amount o f D 0  aqueous sample.  not  drying  observed.  greater the  freeze  l o s s of a c t i v i t y  A n o t h e r method o f c o n c e n t r a t i n g takes  chang-  even under the  temperature, only  2  bromide  c o n d i t i o n s by  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 m e d i a and  favorable  concentrate  AchE s o l u t i o n s were p a s s e d  under v a r i o u s  enough, e s p e c i a l l y a g a i n s t  was  Buffer  c o l u m n t o remove d e c a m e t h o n i u m  then f r e e z e d r i e d twice  ing the  A c h E i n t o D.,0  f r e e z e d r y i n g method i s commonly u s e d t o  enzymes i n t o D 0  and  -  47  C h i c a g o Mark V l i q u i d  possible to concentrate  solu-  i n Secaqueous  during  i n each  the  the  fraction  scintillation  t h e enzyme i n t o  the  -  D0 2  buffer without  buffer  denaturation  solutions.  tribution  -  48  or m i x i n g  but  had  been c o n c e n t r a t e d  of the  tube,  of the  t u b e y e t , where i t w o u l d g e t  III-3.)  n o t y e t been p r e s s e d  tube w i t h s a l t s , sucrose t i o n down d i d n o t  s o l v e the problem.  against  at the  i)  Preparation of E s e r o l i n e  and  MODIFIED ENZYME AND  ITS  the  the  sedimenta-  o f e s e r i n e was  sulfate  hydrolyzed  g) was  by  of  i n a 10%  atmosphere.  aqueous r e a c t i o n m i x t u r e  The  extracted with ether o v e r MgSO^ and  f o r 12 h o u r s . 2  The  was  under a  Eserine  stream.  petroleum ether  nitrogen  l a y e r was  Evaporation  dried  of  recrystallized and  at  continuously  organic  g a v e t h e c r u d e e s e r o l i n e , w h i c h was o f b e n z e n e and  (90).  NaOH s o l u t i o n (5 mS,)  f i l t e r e d under a N  prepared  s t i r r i n g i t for 6 hours  room t e m p e r a t u r e  washed w i t h  was  s t o r e d under n i t r o g e n  = 113-115°C, MS:  M  +  =  218  i n the  freezer.  the  from petro-  A l l s o l v e n t s were p u r g e d w i t h n i t r o g e n b e f o r e  the p r o d u c t m.p.  to  VERIFICATION  t o t h e method u s e d by E l l i s  and  stick  Moreover the presence  i n a manner s i m i l a r  leum e t h e r .  bottom  bottom of  t o slow the  E s e r o l i n e , the h y d r o l y s i s product  a mixture  the  bottom  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 .  PREPARATION OF  ether  dis-  However i t  pelletized  e t c . i n order  D.  (0.5  2  w e l l enough a t t h e  Changing of the d e n s i t y g r a d i e n t  a convection  H0  t o s t o p t h e c e n t r i f u g e e x a c t l y a t t h e moment  when t h e enzyme had  tube.  and  2  (A t y p i c a l p r o f i l e o f enzyme a c t i v i t y  i n a t u b e i s shown i n F i g u r e  proved d i f f i c u l t  of the D 0  use  - 49 -  10  20 Fraction  Figure  111-3:  Number  An enzyme d i s t r i b u t i o n Activities  of  30  i n a tube  after  centrifugation.  A c h E and r a d i o a c t i v e c o u n t s  of  tritium,  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  plotted  a centrifugation  The  against  small  tube.  It  number  the of  f r a c t i o n corresponds  i n d i c a t e s AchE i s c o n c e n t r a t e d  the c e n t r i f u g a t i o n mixing  p o s i t i o n of  s o l u t i o n of  into  the  D90  tube without layer.  to at  the the  AchE,  are  tube. bottom  bottom  s i g n i f i c a t amount  of  of of H^O  a  - 50  ii)  Sulfonylation  of  -  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 of AchE, s i n c e  i t i s an  The  too  reactive  fic  sulfonylation  exclusively  in base-catalyzed hydrolysis of  enzyme d o e s n o t  reactive  go  be  was  10  lected. small  and  the  S i n c e the  amounts o f  through further  fluoride MS  : M+  (bp  leads to  c o l d water  (94).  Also  indicating  70%  a  a q u e o u s KF  solution  124-125°C was  col-  the  organic  presence  layer  gave p u r e  of  was  in order to hydrolyze  Redistillation more IR  nonspeci-  follows:  indicated the  residual  methanesulfonyl  a b s o r p t i o n s at  s o l u t i o n o f MeSC^F was  yJi o f Me'SC^F i n 5 ml the  g of  sulfonyl chloride,  125°C, no  and  is  1165  and  965  cm  = 98) .  A stock  be  30  site  sulfonyl chloride  p r e p a r e d as  IR-spectrum s t i l l  starting material.  esteratic  an  modification.  f r a c t i o n b o i l i n g at  t h o r o u g h l y washed w i t h  produces  completely reactivated,  g o f M e S C ^ C l and  distilled  and  groups i n p r o t e i n s  M e t h a n e s u l f o n y l f l u o r i d e was mixture of  sulfonylation  at the  more r e a d i l y a v a i l a b l e  m e t h a n e s u l f o n y l A c h E can the  the  irreversible inhibitor  enzyme w h i c h i s s u l f o n y l a t e d (76,91,92,93).  used f o r  of  isopropanol  l e a s t damaging t o the  addition  of  isopropanol  (up  dissolving  (isopropanol  i s known  enzyme and t o 2%)  d e c r e a s e the  activity).  A small  was  added t o t h e  solution  11S  s e c o n d a l i q u o t , a m o u n t i n g t o an  i t was  t o the  not  of  p r e p a r e d by  to  found that  the  enzyme s o l u t i o n  did  aliquot  A c h E and  200  of  after  a p p r o x . 500  this two  solution hours a  f o l d molar  excess  ,  - 51  o f MeSG^F o v e r and  AchE.  The  -  mixture  was  t h e a c t i v i t i e s were m o n i t o r e d .  solution  kept  After  a t room 6 hrs the  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  h i b i t e d enzyme d i d n o t  recover  temperature  i t s activity  enzyme  and  the i n -  for at least  four  days. The  m o d i f i e d enzyme was  the Amicon f i l t e r and  concentrated  into D 0  buffer with  2  as d e s c r i b e d i n S e c t i o n C - i .  Excess  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 .  pared  s o l u t i o n s were u s e d f o r NMR  experiments  MeS0 F 2  The  within a  prefew  days. E.  S T A B I L I T Y OF In order  THE  ENZYME  to assure  reliability  of the o b t a i n e d  results,  t h e s t a b i l i t y o f t h e enzyme t o w a r d s t h e a p p l i e d s t o r a g e experimental  4°C,  c o n d i t i o n s was  1)  After  storage  11S  AchE s o l u t i o n was  zyme were c h e c k e d . sucrose 2) low  month i n 4 M N a C l b u f f e r a t  diluted activity  into standard and  ( U n i f o r m i t y was  gradient sedimentation  ( 9 5 ) , and  1 M  NaCl  u n i f o r m i t y of the  c h e c k e d by  en-  isokinetic  as d e s c r i b e d i n S e c t i o n  I t i s known t h a t A c h E t e n d s  s a l t media  tail  checked.  f o r one  c h r o m a t o g r a p h y b u f f e r and  and  t o form aggregates  B-ii.) in  (13,21) e v e n i n a f o r m w h i c h d o e s n o t c o n t a i n a i t was  in  t h e 0.1  An  a u t h e n t i c sample  t h e r e f o r e t e s t e d whether t h i s  M NaCl-D 0-buffer 2  u s e d f o r t h e NMR  ( c a . 18 umole A c h E i n D 0 o  occurred  measurements. buffer)  was  - 52 -  tested  f o r u n i f o r m i t y by i s o k i n e t i c  tation  a f t e r exposure  sucrose g r a d i e n t sedimen-  to experimental conditions.  (Sedimenta-  tion  t e s t a s i n S e c t i o n B - i i ; g r a d i e n t was p r e p a r e d  NaCl  buffer  instead o f 1 M NaCl  s o l u t i o n was l o a d e d w i t h o u t 3)  w i t h 0.1 M  b u f f e r , h o w e v e r , and t h e A c h E  dilution.)  To t e s t t h e s t a b i l i t y  o f t h e enzyme u n d e r  t a l c o n d i t i o n s an NMR t u b e was l e f t  experimen-  i n t h e m a c h i n e f o r 24 h r s .  S m a l l a l i q u o t s were t a k e n p e r i o d i c a l l y  and c h e c k e d  for their  activity. F.  NMR MEASUREMENTS All  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  spectrometer (for  a n d / o r a 270 MHz PMR-FT s p e c t r o m e t e r .  XL100) o r 500 y£ ( f o r 270 MHz s p e c t r o m e t e r )  AchE s o l u t i o n were m i x e d w i t h a s m a l l v o l u m e  o f the lis  (~35u£) o f D 0 0  b u f f e r , c o n t a i n i n g t h e d e s i r e d amount o f i n h i b i t o r 3-trimethylsilylpropionate-2,2,3,3-d^ 5 mm NMR t u b e s .  After  p r o b e i t was n e c e s s a r y  (TSP) and f i l l e d  t o w a i t 30-45 m i n u t e s  o f t h e TSP p e a k .  until  the temper-  a p e r i o d of f i v e minutes  checked  when  f o r the  Then t h e h o m o g e n e i t y o f t h e magnet  a d j u s t e d s o t h a t t h e w i d t h o f t h e TSP peak was l e s s  0.8 H z .  into  T h i s was assumed t o be t h e c a s e  AV was l e s s t h a n 0.1 Hz o v e r  was  and s o d i u m  i n s e r t i o n o f t h e s a m p l e s i n t o t h e NMR  a t u r e was e q u i l i b r a t e d .  position  300 u£  than  P o s i t i o n a n d peak w i d t h o f t h e TSP s i g n a l were a g a i n after  e a c h measurement t o e n s u r e  the s t a b i l i t y  o f the  - 53  magnetic either  field  d u r i n g the a c q u i s i t i o n of the s i g n a l .  a shift  broadening  i n TSP  o f t h e TSP  the spectrum  -  peak p o s i t i o n o f more t h a n 0.2 peak w i d t h o f more t h a n 0.2  Hz o r a  Hz was  found,  was d i s c a r d e d .  To c h a n g e t h e c o n c e n t r a t i o n o f an amount o f a s t o c k s o l u t i o n was tube.  Whenever  inhibitor, a  added d i r e c t l y  M e a s u r e m e n t s were resumed a f t e r  i n t o the  temperature  and  r e a d j u s t m e n t o f t h e h o m o g e n e i t y o f t h e magnet.  all  inhibitors  i n neat D 0  b u f f e r were r e c o r d e d  2  certain NMR  equilibrium Spectra for  separately.  ( 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 relatively in  the v i c i n i t y  l a r g e peak f r o m r e s i d u a l w a t e r , w h i c h  o f t h e peak o f i n t e r e s t , c o u l d be d e c r e a s e d  a m p l i t u d e by use o f a band r e j e c t filter let  (96).  Two  filters  o f t h e XL100 e x t e r n a l  s p e c t r a l w i d t h was  filter  were c o n n e c t e d filter  and  l e s s t h a n 100  Hz.  and  a 100  in series  Hz  pass  t o the  out-  c o u l d be u s e d w h e n e v e r T h i s had  the e f f e c t  the dynamic range  capacity.  ( F i l t e r s were p r e p a r e d by t h e d e p a r t m e n t a l  to  T y p i c a l NMR  270 MHz  Spectrum (5.12 usee  of  were  electronic  (Figures in brackets refer  spectrometer):  sec), Pulse delay = 0  (200 H z ) , A c q u i s i t i o n  time = 5 sec  (393.22 m s e c ) , P u l s e w i d t h = 25  u s e e ) , S e n s i t i v i t y enhancement = 3 sec  n i n g r a t e = 35-40 c p s , T e m p e r a t u r e = 17-30°C transitions  the  o f t h e XL100 c o m p u t e r memory  parameters  w i d t h = 100-130 Hz  (6.50  in  low  increasing  shop.)  appeared  = 300-1000 s c a n s .  (none), S p i n -  (io°C), Number o f  -  54 -  CHAPTER I V RESULTS  A.  STABILITY As  a  AND  reported  specific  PURITY  in Section  activity p e r mg  AchE which  hydrolyzes  above  choline  choline  present  enzyme  The  form  a r e shown  decreased  stable  under  To in  while  test  t h e NMR  jected IV-2,  form  1 month IV-1.  showed  i s a single  of  Since  acetyl-  of  11S A c h E  100%  acetyl-  with  this  shows  the enzymic  Therefore,  o f 11S  a t 4°C i n 4 M  figure  lis  the  pure.  due t o a u t o l y s i s  that  the  reported  i s 10.8 m m o l e s  storage  a t t h e enzyme  a concentrated  gradient  (23,24),  that the during  activity  AchE  NaCl  seems  had t o be  conditions.  f o r aggregation  experiments,  per min  centrifugation  This  has n o t changed  storage  p e r min would  by e l e c t r o p h o r e s i s ( 1 8 ) ,  gradient  significantly. these  o f AchE  t h e r e f o r e be a l m o s t  an a s s a y  to sucrose there  single  i n Figure  t h e amount o f  t o 10.34 mmoles  (13,18,96).  and a f t e r  o f t h e enzyme  storage,  mg  o f sucrose  before  Since  C u r r e n t l y , t h e maximum  activity  enzyme had  acetylthiocholine  of acetylcholine  corresponds  min.  shows  must  results  taken  buffer  not  activity  of  of acetylthiocholine  hydrolyzed/min,  specific  AchE  specific  the p u r i f i e d  9 mM  per min.  1 ymole  activity  hydrolyzed/mg  reproducible  III-A-ii-b,  of protein  0.87 y m o l e s  specific  ENZYME  of approximately  hydrolized  hydrolize  OF T H E  NMR  centrifugation.  symmetric  peak  concentration sample  was  A s shown  corresponding  used  subi n Figure t o t h e 11S  - 55 -  Figure  IV - I :  Sucrose gradient and a f t e r  Figure  I V -2:  (b)  the  Sucrose gradient w h i c h was  kept  profile storage  of  ||S  AchE b e f o r e  i n 4M NaCl  p r o f i l e of  baffer  concentrated  i n t h e NMR e x p e r i m e n t a l  IIS  (a) at  and 4*C.  AchE  conditions.  -  form.  -  56  I f t h e enzyme were a g g r e g a t e d one w o u l d e x p e c t t o see  a peak skewed t o w a r d s t h e l e f t due  t o t h e p r e s e n c e 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 t h e enzyme d o e s n o t a g g r e g a t e u n d e r d i t i o n s o f t h e NMR Finally,  experiment.  t o e n s u r e t h a t t h e enzyme r e m a i n e d  t h e e x p e r i m e n t i t was d a y a t 17°C  and  experiment. buffer B.  a t 4°C  assayed f o r a c t i v i t y  12 h r s a t 30°C u n d e r  Also,  the con-  after  active  during  storage for a  the c o n d i t i o n of the  t h e enzyme a p p e a r e d  t o be s t a b l e  in  D0 2  f o r up t o 1 week.  INHIBITORS WITH UNMODIFIED ENZYME The  *H NMR  spectra of atropine s u l f a t e  trimethylammonium chloride  (PTA)  chloride  i n 0.1  (TMA)  M NaCl D 0 2  and  (see F i g u r e  IV-3),  phenyltrimethylammonium  buffer  s e n c e o r a b s e n c e o f A c h E , were e x a m i n e d  (pH 7.0)  i n the p r e -  to investigate  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 t h e enzyme.  the  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 , w h i c h r e s o n a t e a t 2.68 for  NMR  a t r o p i n e , a t 2.94  downfield  f r o m TSP  ppm  f o r TMA,  and a t 3.66  peak r e s p e c t i v e l y .  c h a n g e by more t h a n 0.5  ppm  f o r PTA  Their p o s i t i o n s d i d not  Hz upon a d d i t i o n o f A c h E .  s p e c t r a u s e d t o m e a s u r e l i n e w i d t h s had  A l l the  a specral width of  Hz o r 110 Hz d e p e n d i n g on t h e l o c a t i o n o f o t h e r p e a k s i n t o the d i s p l a y e d s p e c t r a l frequency range. e a c h CH^  peak was  measured  ppm  at h a l f maximal  The  100  folded  linewidth  h e i g h t from a  of  line  -  57  -  cis-2,6-dimethylspiro(piperidine-1,1-pyrrolidium)  Figure  IV-3:  Structure NMR  of  acetylcholinesterase  experiments'.  inhibitors  bromide  used  in  - 58  drawn b e t w e e n t h e An  accuracy  widths  of  flat  b a s e l i n e s on e i t h e r s i d e o f t h e  ~ ± 0.05  with a ruler.  the g r e a t e s t source  -  Hz  can  be  achieved  This technique  by m e a s u r i n g  of l i n e w i d t h e r r o r s , s i n c e the  most e x t r e m e p o s s i b l e v a l u e s  inhibitor reported The  p e a k s , d e p e n d i n g on  NMR  for l i n e w i d t h s of  b a s e l i n e s are drawn,  a b s e n c e o f t h e enzyme a r e  w i t h the  s p e c t r a o f TSP  inhibitor  f o r a t r o p i n e , PTA  hibitor  uneven b a s e l i n e .  s p e c t r a of the methyl group i n a t r o p i n e  l a t i o n of the width  the  accumula-  are  as e r r o r s .  p r e s e n c e and together  obtained  how  peak  of drawing b a s e l i n e s i s  t i o n o f a l a r g e number o f s i g n a l s g i v e s an The  peak.  concentrations  taken  signals. and are  TMA  in  shown i n F i g u r e before  and  tabulated  with  IV-4»  after  Observed changes i n  along  the  accumuline-  t h e enzyme and  i n Table IV-1.  AAy  inis  the  s y m b o l u s e d f o r t h e b r o a d e n i n g o f t h e m e t h y l peak due  to  the  i n t e r a c t i o n of  in  AAy  Hz.  inhibitor  i s c a l c u l a t e d by  b e t w e e n a m e t h y l peak and enzyme and  w i t h enzyme and  i s expressed  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 a TSP  peak i n t h e  absence of  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  o f t h e enzyme.  Here the v a r i a t i o n  i n the  d u r i n g measurement.  I t was  not  necessary  b a s e l i n e a d j u s t m e n t i n t o c o n s i d e r a t i o n f o r TSP an  ideal  terase  flat  baseline.  i s reported  The  concentration  presence  l i n e w i d t h of the  peak i n d i c a t e s t h e change i n t h e h o m o g e n e i t y o f t h e field  the  TSP  magnetic  to take p e a k , as  the i t had  of a c e t y l c h o l i n e s -  as t h e 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 ,  taking  -  Figure  IV-4:  The  59  -  'H NMR s p e c t r a o f  The m e t h y l  peaks o f  a r e shown t o g e t h e r right  hand s i d e ,  accumulation of  atropine  sulfate with  atropine with with  the  (B)  r e f e r e n c e TSP  spectra.  The  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 atropine  and w i t h o u t  w h i c h were t a k e n b e f o r e atropine  r e s o n a n c e and by 0 . 2 0  times  unmodified  the  and a f t e r  the  times for  (A)  p e a k s on  original  AchE.  100Hz  for  t h e TSP  the resonance.  Table  IV-1  C o n c e n t r a t i o n i n h i b i t o r  ;(mM)  a c e t y i c h o l i n e s t e r a s e (yM)  Temperature  C °  Observed  l i n e w i d t h  (Hz) i n h i b i t o r  TSP  0.3  ~  30  1.0±0.1  0.5  0.3  15  30  1.4±0.1  0.8  0.5  —  30  0.6  0.5±0.1  30  0.8±0.1  0.7±0.1  19  0.6  0.6  a t r o p i n e  T r i m e t h y l ammonium c h l o r ide (TMA)  P h e n y m e t h y ammon c h l o r (PTA)  l t r i l ium i d e  Broadening due t o b i n d i n g AAy (Hz)  0.1+0.14  0.0±0.2 0.5  14.6  0.8  0.1±0.14 0.8  15  19  0.9±0.1  0.8±0.1  - 61  -  80,000 as t h e m o l e c u l a r w e i g h t f o r an a c t i v e s u b u n i t . all  t h e enzyme u s e d  i n the experiment  tical  in catalytic  s i n c e each u n i t  activity  and  lis,  t a k e s the form of  which c o n s i s t s of four a c t i v e s u b u n i t s , each c o n s i d e r e d as i n d e p e n d e n t  Although  active  subunit i s  i s known t o be  iden-  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 ) . It line  i s clear  broadening  The  small line  PTA  are smaller  possible or  f r o m T a b l e I V - 1 t h a t t h e r e i s no (AAy)  observed  broadenings  (0.1 Hz)  observed  three.inhibitors. for atropine  than e x p e r i m e n t a l u n c e r t a i n t y ,  t o o b s e r v e any  line  at higher concentrations.  since Kato  f o r any o f t h e  broadening at t h i s c o n c e n t r a t i o n These r e s u l t s a r e  unexpected  (71-74) o b s e r v e d a c o n s i d e r a b l e l i n e  broadening  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  Tjjr- i s d e p e n d e n t on f c o n c e n t r a t i o n . One ening bind in  B  inhibitor,  w h i c h becomes s m a l l e r  no b r o a d e n i n g .  S e c t i o n A t h a t t h e enzyme u s e d active  observe  any  first  broaden-  at a higher  inhibitor  e x p l a n a t i o n of the p r e s e n t absence o f  giving  and  However i t was  broadcannot  proved  i n t h e s e e x p e r i m e n t s was  t h r o u g h o u t , so t h i s  at  i n T a b l e I I - l , where  c a n n o t be t h e  T h e r e a r e two o t h e r p o s s i b l e c a s e s where one  The  line  i s t h a t t h e enzyme i s d e n a t u r e d o r a g g r e g a t e d  f o r m and  and  so i t i s n o 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 ing  significant  uni-  cause. would  not  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 .  case  1 r e g i o n and ^ — 2 2B  i s when t h e s y s t e m > >  P  B  A  2 '  w  n  e  r  e  i s i n the slow exchange  the observed  linewidth i s  - 62 -  described according t o the equation given as  i n Table  I I - l and  follows:  1  =J L  T  T  2  +  f  P  B BA  2 A  If the binding constant  (K^) f o r t h e i n h i b i t o r ,  concentration of the i n h i b i t o r tion  (I )  and t h e enzyme  Q  ( E l a r e known, t h e f r a c t i o n  the i n i t i a l  o f bound i n h i b i t o r  o  (f„) c a n a  be c a l c u l a t e d  by s o l v i n g  the following quadratic equation  x  K  forx  D  where x i s t h e c o n c e n t r a t i o n o f t h e bound i n h i b i t o r ~. ° all  concentra-  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 a r e 1 1 1 - 2 % . When — >> P_ i s assumed and t h e v a l u e f o r 2B 2B i s known, t h e p o s s i b l e r a n g e f o r P c a n be e s t i m a t e d . The BA K  T  B  A  T  R A  maximum p o s s i b l e v a l u e o f =1— f o r a m e t h y l g r o u p a t t a c h e d t o a 2B 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 f r o m F i g u r e I I - l f o r a known X  rotational correlation in the present  time  T .  F o r t h e 11S f o r m o f A c h E u s e d  study, the molecular  weight  partial these  s p e c i f i c volume  values  s e c , and ~ (AAy)  clue  t  = 400 Hz f r o m F i g u r e I I - l . i n t e r a c t i o n of i n h i b i t o r  o  TT 1 2  »  P  line B A  broadening  as f o l l o w s  Substituting  6 i n the t h e o r e t i c a l s e c t i o n together T  (n) o f 0.01 f o r AchE s o l u t i o n ,  f e r e n c e b e t w e e n —^— and expected  (v) i s 0.72 cm /g ( 2 9 ) .  into equation  with a v i s c o s i t y  i s 320,000 and t h e  3  -  The l i n e  w i t h enzyme  — , which i s f„P .. ^ 2A D  i s estimated  = 1 x IO  - 7  broadening i s the d i f -  Hence t h e maximum  from t h e r e l a t i o n s h i p  -  7TT  *  2 B  L  "  B  1  TTT .*. and  AAy  l i n e  <  400  8  Hz  AAy  <  broadening  slow,  since The  system l i n e  i n  the  and  the  =  _L_  "  TTT  0.1  f „  i s 1  v i s i b l e  maximum  no  f a s t  i f  R  i s  400  Hz.  If  or  the  methyl  w i l l  be  s m a l l e r .  l i n e  broadening  exchange  of  the  broadening  NMR  i s  a c c o r d i n g  rate  to  i s  t h i s  i s  when  Hence Table  no  i s  s p e c t r a  seen  r e g i o n .  0.1  Hz.  the  the  expected  I I - l ,  as  2 B  value  fp,-7jr—  the  i n h i b i t o r  can  take  i s  8  Hz  when  2B  B l T i  2B zyme  Hence,  1  BTTT  p o s s i b l e  —=—  exchange.  the  exchange  f  -  slow  r e s o l u t i o n  when  = 2 A  0.02  very  e x p r e s s e d ,  TTT  =  B  for  be  very  ~  2  Hz  case  broadening  A A  and  maximum  second  i s  Hz  w i l l  the  -  BA  2 B  <<  perhaps  TT B  63  i s  l o o s e l y  bound  to  the  e n -  t t T  s i b l e very  not f a s t  to  has  coupled  i f  worthwhile  i n h i b i t o r  methyl  on  be  Exact  1  to  n o t i c i n g peak,  0.1  s i t e  which 0.5  of  hence  m u l t i p l e t  i s  the  5  mM  Hz,  f r e e l y , the  i f  the  very  of  i s  compound,  pos-  i s  f l e x i b l e .  in It  though  i t  broadening  at  c i s - 2 , 6 - d i m e t h y l s p i r o -  ( s t r u c t u r e  TJT—, 2B  i t  system  s i g n i f i c a n t was  —=—  expected  Therefore  only  gave  s t r u c t u r e  s p i n s  Hz.  broadening  that  of  than  - p y r r o l i d i u m ) b r o m i d e  v a l u e s  the  l e s s  l a b e l e d  2  due  i n h i b i t o r  than  l i n e  l  ever  i s  l e s s  any the  the  —=— 7 T T 2B  c o n c e n t r a t i o n  ( p i p e r i d i n e - 1 , 1 I V - 3 ) .  w i l l  r e g i o n  be  an  When  observe  might a  group  are the  not  shown  in  o b t a i n a b l e  peaks.  Figure how-  -  C.  ESERINE  The enzyme  b i n d i n g  was  C-methyl TSP  group,  s o l u t i o n  The  The t o  B,  w i t h i n  be  r e g i o n or  resonances i n  i s  and unbound than  1  Hz  bound  g r e a t e r  f i n d i n g  enzymes  do  e x c e p t i o n  s h i e l d i n g  the  the l i n e  weighted  s p e c i e s  and f r e e  n o t  phenyl  f o r  t h e  changes  near  the  l e s s  i s  i n i s  t h e  I V - 5 .  (pH  7.0)  i n  a r e  However,  of  1%),  these  no l i n e s  groups  expected  t o  the  i n  must  p o s i t i o n  f o r  a  s h i f t  c h e m i c a l be  s h i f t  s i t e  i s t h e  l e s s  (97).  t h e of  s h i f t  other  bound.  has a In  50  bound.  of  when  which  f a s t  than  a r e  i n h i b i t o r s  lysozyme  t h i s  t o  m o l e c u l e s  c h e m i c a l  r e g i o n  i n  rate  move  because  most  b i n d i n g  exchange  exchange  of  i n h i b i t o r  of  Figure  than  slow  d i f f e r e n c e  i n  as  30°C  expected  s i n c e  i n  stock  AchE.  However,  methyl  from  buffer  enzyme.  be  of  f i e l d  s u l f a t e  frequency  average  the  i n h i b i t o r s  group  t o  If  i s  ( 8 2 ) .  s u r p r i s i n g  produce i s  of  a t  the  of  B.  l i n e w i d t h  shown  broadening  p o s i t i o n  2%  of  carbamylated  same  e s e r i n e  system  i n  e s e r i n e  the  resonance  S e c t i o n  peak  than  i s  n o t  that  i n  of  t o  ppm down  s p e c t r a  presence  no  of  (estimated  the  t h e  change  i n  presence  was o b s e r v e d ,  Hz  when  i n  s i n c e  the  which  t h e  e r r o r  i n  d e s c r i b e d  f a s t ,  t y p i c a l  IV-3)  1.51  a d d i t i o n  s o l u t i o n ,  t h e  changes  t h e  The  a t  group  between  This  r e s o n a t e s  p o s s i b i l i t y  frequency  l e s s  which  observed  as  very  bound  the  enzyme  e x c l u d e d  (see F i g u r e  measuring  e x p e r i m e n t a l  be  ENZYME  by  broadened  The can  e s e r i n e  produced  s i g n i f i c a n t c o u l d  of  s e q u e n t i a l  the  C-methyl  markedly  CARBAMYLATED  s t u d i e d  peak.  Section  WITH  -  64  d e the  f a s t  -  Figure  IV-5:  The  'H  NMR s p e c t r a o f  C-methyl of  peaks o f  Also  i n the  and a f t e r original  (last  eserine sulfate with  of  bottom  shown a r e t h e  -  e s e r i n e a r e shown  increasing ratio  spectra  65  i n the  carbamylated first  e s e r i n e t o AchE from top row w e r e o b t a i n e d  r e f e r e n c e TSP  column)  peaks  column i n to  taken  before  The AchE.  (second  eserine spectra.  been r e d u c e d 0 . 2 6  times.  The  order  bottom.  i n the absence of  the accumulation of  120Hz w i d e s p e c t r a h a v e  AchE.  column) The  - 66 -  e x c h a n g e c a s e P , >> Aio a s g i v e n  i n Table  n  II-l.  I t follows f Aa) —5 (Table 2  B  that  i n t h i s region the expected  II-2)  i s much l e s s  t h a n fgAco.  line  broadening  ALO i s c a l c u l a t e d t o  However  be l e s s t h a n 50 Hz and f _ i s 2%.  BA  Therefore  i t c a n be s a i d  t h a t i f t h e exchange r a t e i s f a s t the l i n e broadening much s m a l l e r t h a n expects  1 Hz and w o u l d n o t be o b s e r v a b l e .  t o s e e no l i n e  broadening  w o u l d be S i n c e one  when t h e s y s t e m i s i n t h e  slow or f a s t exchange r e g i o n s , the l i n e for  e s e r i n e must be c o n t r o l l e d  by t h e T  and  t h e e x c h a n g e r a t e must be v e r y  broadening  observed  relaxation  2 B  process  fast.  T h e r e a r e two methods t o d e d u c e t h e v a l u e o f =r- f o r t h e bound i n h i b i t o r region.  when 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  A s m e n t i o n e d i n t h e t h e o r y s e c t i o n b o t h methods  disadvantages.  have  I n o r d e r t o use t h e e x p r e s s i o n by G e r i g  (Equation 7 ) ; AAy =  I  o  E °  D  • Ay  (9)  E I  t h e v a l u e o f K_ must be known. D  replaced  by AAy  a n (  3 Ay  E I  •  Here  Although  (7^T  2  T  TT  2 A  and  — are  TTT  2 B  for eserine binding to  -6 AchE  (3.3 x 10  experiment,  M)  (46) i s much s m a l l e r t h a n  t h i s value  i s expected  I  t o be l a r g e r  i n the present f o r carbamylated  AchE and c a n n o t s i m p l y be assumed t o be n e g l i g i b l e with  I  when an e x a c t v a l u e o f K  D  f o r carbamylated  known.  I n t h e o t h e r m e t h o d , t h e v a l u e o f 7^-  hibitor  may be o b t a i n e d w i t h o u t  using  compared AchE i s n o t  f o r t h e bound i n -  k n o w i n g t h e v a l u e o f K , by  i n s t e a d t h e e x p r e s s i o n by K a t o ( E q u a t i o n  D  8)  - 67 -  J  oo = oo^AAy F - n E  K  ( 1 0 )  u  However t h e a s s u m p t i o n made t o d e r i v e t h i s e q u a t i o n ; >> •—-, may n o t be r e a s o n a b l e  1  m  ' '  m  The o b s e r v e d  l i n e broadenings  i n the present  according  case.  ( A A Y = "Tfr ~ ~ ^ — ) -  TTT  plotted  namely  t o both equations  TTT  2  a r  ^  2 B  as seen i n F i g u r e  I V - 6 and  _g  Figure  IV-7. A K  D  value  o f 3.3 x 10  AchE i s used t o o b t a i n F i g u r e calculation  o f AAy  a n <  Figure  binding to  I V - 6 . The same m e t h o d s f o r t h e  3 t h e e v a l u a t i o n o f t h e 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 a r e used here. straight  M for eserine  l i n e s obtained  From t h e s l o p e s o f b e s t  fitting  by w e i g h t e d * l e a s t s q u a r e f i t i n  I V - 6 and I V - 7 , t h e l i n e w i d t h s o f a C - m e t h y l peak o f  e s e r i n e bound t o t h e c a r b a m y l a t e d AchE a r e d e t e r m i n e d 273 + 23 Hz and 248 + 29 Hz r e s p e c t i v e l y . is  the standard  plotted  The v a l u e s  agree w i t h i n e x p e r i m e n t a l from F i g u r e  o f AyEI obtained  errors.  gives  represents  here  through  f r o m two methods  The i n t e r c e p t o f t h e s l o p e  I V - 6 i s c a l c u l a t e d t o be 0.0 ± 0.1 and s o t h e  s t r a i g h t l i n e goes through the o r i g i n IV-7  The e r r o r n o t e  d e v i a t i o n of s t r a i g h t l i n e s going  points.  t o be  as e x p e c t e d .  Figure  t h e c a l c u l a t e d i n t e r c e p t o f -115 ± 168 yM, w h i c h the K  Q  value  of eserine  interacting with  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 - m i n v a l u e . 30 '  (  carbamylated  -  Figure  IV-6:  A plot with  Figure  IV-7:  MiV for  respect to  A plot of  of  of  the  IV-6  the  -  C-methyl  varying  and  IV-7  from the observed  strength,  pH  for  the  concentrations were p l o t t e d being the  linewidths  above f i g u r e s  30 C ,  resonances of  eserine  EQ/^-KQ.  a n d 10 r e s p e c t i v e l y , AAV  spectra for  proton  r e c i p r o c a l o f AAV  eserine vs.  Figures  68  7.0.  of  C-methyl  resonances  eserine  sulfate.  according to equations l i n e broadening  as d e s c r i b e d i n t e x t .  were r e c o r d e d a t  100MHz  deduced All field  9  -  AchE.  69  However, as a n t i c i p a t e d  calculated value  from Chapter  I I I , the i n t e r c e p t  ( 1 1 5 ± 1 6 8 yM) i s n o t v e r y u s e f u l s i n c e t h e K  i s expected  obtained  -  forAy  E I  t o be s m a l l e r t h a n 1 0 0 yM. '  i t  c  a  be s a i d  n  From t h e v a l u e  that the assumption  made t o  o b t a i n F i g u r e I V - 7 , n a m e l y — = r — >> - ~ — , i s r e a s o n a b l e . 7 T T  K  D  2B  T T T  f r o m one f o r n a t i v e A c h E , a s t h e s t r a i g h t  is  Also  2A  f o r c a r b a m y l a t e d AchE w o u l d n o t have been g r e a t l y  origin  D  different  l i n e goes through the  and t h e a g r e e m e n t o f A y j v a l u e s o b t a i n e d by two methods E  good. When K a t o c a l c u l a t e d  t h e l i n e w i d t h o f 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 w i t h n a t u r a l enzyme  (71,72,73).  inhibitor However,  and was i n t e r a c t i n g  i t h a s 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 b y c a r b a m y l a t ing the c a t a l y t i c ine  serine residue (46,108,  Chapter  i s now w i d e l y a c c e p t e d a s an i r r e v e r s i b l e  AchE w i t h a i n h i b i t i o n M ^ min  I I ) and e s e r -  inhibitor of  r a t e c o n s t a n t k^ v a l u e o f 3 . 3 x 1 0  f o r the reaction k. EH + PX  EP +• XH  I n t h e above r e a c t i o n EH, PX, EP and XH r e p r e s e n t A c h E , 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 eserine)  respectively.  (a h y d r o l y s i s p r o d u c t o f  The t i m e r e q u i r e d  t o carbamylate 9 9 %  o f t h e AchE a t t h e c o n c e n t r a t i o n s o f enzyme and e s e r i n e u s e d in the experiment hence j u s t i f i e d  i s calculated  t o conclude  t o be 1 . 6 x 1 0  5  min.  It is  t h a t a l l the changes observed i n  - 70  t h e NMR  -  l i n e w i d t h o f t h e m e t h y l g r o u p a r e due  t o the  interac-  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 n o t w i t h n a t u r a l A c h E . A l t h o u g h t h e k^ v a l u e f o r e s e r i n e  inhibitor  i s well  e s t a b l i s h e d , t h e r e a r e d i s c r e p a n c i e s among t h e v a l u e s o f t h e r e c o v e r y r a t e o f i n h i b i t e d AchE w i t h Dr. R o u f o g a l i s ) . produce  (46 and p e r s o n a l c o m m u n i c a t i o n  I f t h e enzyme r e c o v e r s e f f i c i e n t l y  enough e s e r o l i n e , t h e m e t h y l peak i n e s e r o l i n e  i n t e r f e r e w i t h t h e one  in eserine.  o f two p e a k s m i g h t a p p e a r  In f a c t the  to produce  T h i s was  studied  (99,100).  acetyllinewidth  In o r d e r t o t e s t  the c h e m i c a l s h i f t s of the methyl groups of e s e r o l i n e  e s e r i n e a r e n e a r enough t o g e t h e r t o p r o d u c e e s e r o l i n e was was  are c l o s e t o -  t h r o u g h t h e c h a n g e i n NMR  of the m e t h y l group of A c e t y l c h o l i n e if  superposition  i n d e e d t h e c a s e when t h e b i n d i n g o f  c h o l i n e t o A c h E was  will  b r o a d e n i n g o f t h e peak  when t h e r e s o n a n c e p o s i t i o n s o f t h e two p e a k s gether.  p r e p a r e d as i n C h a p t e r  o b t a i n e d i n t h e same b u f f e r  binding experiments. t o r e s o n a t e a t 1.46 from the C-methyl possible  ppm  overlapping  I I I and  solution  The C - m e t h y l  to  i t s NMR  as was  and peaks,  spectrum  used  i n the  g r o u p o f e s e r o l i n e was  d o w n f i e l d f r o m TSP,  resonance of e s e r i n e .  a full  5 Hz  away  T h e r e f o r e , i t i s im-  f o r t h e two m e t h y l r e s o n a n c e s t o be s u p e r i m p o s e d  t h e o b s e r v e d l i n e b r o a d e n i n g i s i n d e e d due b e t w e e n e s e r i n e and c a r b a m y l a t e d A c h E .  found  t o the  and  interaction  - 71  P.  INHIBITORS WITH METHANESULFONYLATED AchE The  results  fication the  obtained  i n S e c t i o n C i n d i c a t e t h a t the  o f A c h E a t t h e c a t a l y t i c s i t e a l l o w s one  b r o a d e n i n g o f NMR  p e a k s e i t h e r by a l t e r i n g  r a t e o r t h e bound l i n e w i d t h o f i n h i b i t o r s are  r a t e s from m o d i f i e d faster.  hibitors  AchE f o r t h e s e  enzyme and  m e t h y l peaks f o r the  two  f r e e of methanesulfonyl i n Chapter  in Figure  tation  no  0.2  ± 0.1  are  0.5  IV-8  and of  t e s t e d by  mM  and  when t h e 18.6  uM  with modified  the  can  2  be  w i t h TSP  AchE.  The  are  observed  intertheir  s u l f o n y l a t e d AchE, as  (AAy  observed and  to expec-  modified  However t h e  be  AchE  C-methyl  b r o a d e n when t h e s e  T h i s can  =  inhibitors  seen i n F i g u r e s  IV-9  concentrations  shown. l i n e broadenings  same manner as d e s c r i b e d s y s t e m was  same i n -  PTA)  peak, c o n t r a r y  respectively. indeed  to  s e e n f r o m m e t h y l peaks o f  c o n c e n t r a t i o n o f TMA  do  off  b u f f e r i s prepared  l i n e b r o a d e n i n g was  PTA  the  l i n e w i d t h of  The  in D 0  AchE  are expected  l e t t i n g the  I V - 1 0 , 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 inhibitors  Some r e v e r s i b l e  ( a t r o p i n e , TMA,  comparing  fluoride,  together  p e a k s o f a t r o p i n e and interact  was  enzyme f o r m s .  I I I . As  significant Mz)  exchange  consequently  inhibitors  as u s e d f o r n a t u r a l enzyme  described  to observe  the  inhibitor.  (26,46 ,76,92) and  This hypothesis  act with modified  TMA  the  modi-  known t o b i n d more p o o r l y t o t h e m o d i f i e d  t h a n t o the n a t u r a l AchE  be  -  i n the v e r y  (AAy)  i n S e c t i o n B.  are measured I t was  f a s t exchange r e g i o n  in  the  assumed t h a t  f o r the  the  same r e a s o n  - 72 -  Figure  IY-8:  The  'H NMR s p e c t r a o f  modified AchE. absence with 100  the  (A)  and  trimethylamine  The m e t h y l (B)  of  peaks of  hydrochloride  trimethylammonium  s u l f o n y l a t e d A c h E a r e shown  r e f e r e n c e p e a k s on t h e  with  right  hand s i d e .  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  times.  in  the  together  The  original  - 73 -  Figure  IV-9:  The  H NMR s p e c t r a o f  The m e t h y l in order of  peaks o f  the  left  i n c r e a s i n g r a t i o of  the  i n h i b i t o r to  to  atropine  sulfate with modified  at  AchE f r o m top shown t o  atropine  bottom.  the r i g h t .  All  Corresponding  AchE.  hand s i d e a r e the  r e f e r e n c e TSP  shown  sulfonylated peaks  are  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  200Hz w i d e s p e c t r a r e d u c e d 0 . 2 6  times.  - 74 -  Figure  IV-10:  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  with modified AchE.  The m e t h y l  ammonium  a r e shown t o g e t h e r  TSP  (far  peaks  to bottom  (to  the  right).  i n order of  AchE r a t i o , absence o f  left)  and t h e enzyme.  phenyltrimethylwith  the  .31  times.  top  sulfonylated  s p e c t r a were o b t a i n e d  s p e c t r a shown a b o v e  60Hz w i d e s p e c t r a r e d u c e d  reference  s p e c t r a are arranged from  i n c r e a s i n g i n h i b i t o r to  bottom All  The  peaks o f  in  the  are the o r i g i n a l  - 75 -  as g i v e n  i n Section C i n order  broadening according g r a p h s a r e shown value For K  D  K  to equations  in Figures  = 5.3 x 1 0 ~  D  5  widths  (9) and  (10) .  M f o r PTA b i n d i n g  S e c t i o n C.  e i  The  (10) i s u s e d f o r p l o t t i n g  ) are obtained  PTA  i n t h e same manner  described in  methyl  o f Aygj o b t a i n e d f o r  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  to use e q u a t i o n s zyme) and A y  (9) and  >> Ayj»  E I  a  r  ( 1 0 ) , namely I e  n  be t h e r e a s o n why  l o o k s more l i k e  a hyperbola  o  t  >>K  Q  satisfied. the p l o t t e d  these  graph i n Figure  than a s t r a i g h t  line  i n t e r c e p t , which i m p l i e s a negative  straight  line  i n Figure  K» D  s p e c t r a were o b t a i n e d  by a NIC-270 FT NMR  the ones f o r a t r o p i n e . p e a k s were o b s e r v e d a t 20°C u s i n g decreased observed.  In t h i s  a 270 MHz  spectrometer.  origin. by u s i n g  s e c t i o n most o f t h e spectrometer  The l i n e w i d t h s o f a t r o p i n e  a t 20°C and 30°C u s i n g  IV-13  A l s o the  I V - 1 2 d o e s n o t go t h r o u g h t h e  spectrometer.  condi-  and g i v e s a  I n 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 XL100 NMR  required  (for n a t u r a l en-  D  However  positive  a Varian  line-  They a r e f o u n d t o be 14 ± 1 Hz and 16 + 2 Hz f o r  The a g r e e m e n t o f t h e two v a l u e s  t i o n s may  a  as t h e  The bound  t h e PTA m e t h y l peak and 97 ± 11 Hz f o r t h e a t r o p i n e peak.  plotted  t o n a t u r a l AchE ( 4 6 ) .  f o r a t r o p i n e h a s n o t been m e a s u r e d .  (AY  line  I V - 1 1 , IV-12 and I V - 1 3 u s i n g  atropine only equation value  t o p l o t the observed  except  methyl  a V a r i a n XL100 and  When t h e t e m p e r a t u r e i s  f r o m 30°C t o 20°C, a 0.2 Hz b r o a d e n i n g o f t h e l i n e i s The r e v e r s e  i s expected  i n the slow exchange r e g i o n  -  76 -  E0/AAV(«M/HZ)  Figure  IV-11:  A plot  o f t h e r e c i p r o c a l o f LhV  resonances  with  respect  atropine  sulfate  recorded  a t 100MHz f i e l d  points  were  to varying  (IQ).A  obtained  f o r the methyl  from  concentrations  corresponds strength, spectra  group  to the  20°C.  spectrum  The  obtained  o^  remaining  a t 270MHz,  20°C.  Figure  Iv-12:  A plot  o f LLV  f o r the methyl  trimethylammoniurn w i t h Figure  IV-13:  proton  respect to  resonances o f phenyl -  EQ/^Q-KQ.  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 of  phenyltrimethylammoniurn  Figures  IV-12  vs.  group  resonances  concentration.  and I V - 1 3 were p l o t t e d  according to  equations  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 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.  were  - 78 -  since the decrease (P  B A  )  i n temperature  lower the o f f r a t e  i n t h e 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  the l i n e broadening with L j f i t well tained in  will  observed  as  by t h e 270 MHz  a t 20°C w i t h XL100  Also  (indicated  the s t r a i g h t l i n e o f F i g u r e IV-11 obspectrometer.  the f a s t r e g i o n the l i n e  I f the exchange r a t e i s  b r o a d e n i n g due t o t h e i n t e r a c t i o n i s  given a c c o r d i n g t o the equation i n Table I I - l AA  1/1 ^ 2 i  The c h a n g e will  i n magnetic  decrease  system  was  __1 \ _ -F (Acof 2A BA B T r ±  field  s t r e n g t h f r o m 270 MHz  Aco and h e n c e AAy by 1/7.3  Hz  Hence i f t h e  i n the f a s t exchange r e g i o n , the v a l u e of E /AAy, o  o b t a i n e d w i t h a X L 1 0 0 , w o u l d have been o n e s o b t a i n e d w i t h 270 MHz further  times.  t o 100  t o the r i g h t from  7.3 t i m e s g r e a t e r t h a n  s p e c t r o m e t e r and w o u l d be straight line.  t i o n s c o n f i r m t h e s p e c u l a t i o n made e a r l i e r r a t e c a n n o t be e i t h e r  located  These two o b s e r v a t h a t the exchange  i n the slow or the f a s t exchange  region.  - 79 -  CHAPTER V DISCUSSION  A.  INHIBITORS WITH UNMODIFIED A c h E Experiments  similar  t o those d e s c r i b e d i n Chapter  I V were  c a r r i e d o u t by K a t o , and showed c o n s i d e r a b l e b r o a d e n i n g f o r b o t h t h e a t r o p i n e and e s e r i n e p e a k s inhibitor work  i n t h e p r e s e n c e o f A c h E , even a t  c o n c e n t r a t i o n s 100 t i m e s h i g h e r t h a n i n t h e p r e s e n t  (71,72,74).  K a t o ' s v a l u e s o b t a i n e d f o r t h e bound  line-  w i d t h o f b o t h t h e a t r o p i n e N - m e t h y l and e s e r i n e C - m e t h y l  peaks  a r e i n c o n s i s t e n t , b e i n g 2 1 , 0 0 0 , 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 a r e a l l t o o l a r g e , as  linewidths f o r small molecules t i g h t l y not exceed  bound t o 11S AchE s h o u l d  400 H z , a s e s t i m a t e d i n C h a p t e r  IV.  To c l a r i f y  t h e e x p e r i m e n t was r e p e a t e d u s i n g t h e same i n h i b i t o r s 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 expected.  Our r e s u l t s  line  this,  as K a t o .  b r o a d e n i n g were  i n d i c a t e d no t r a c e o f l i n e  broadening  with a l l i n h i b i t o r s except cis-2,6-Dimethylspiro-(piper i d i n e 1,1'-pyrrolidium)bromide.  The e x c h a n g e r a t e c a n be i n e i t h e r  the slow or t h e f a s t exchange r e g i o n b u t t h e c h e m i c a l d i f f e r e n c e b e t w e e n bound and f r e e w i t h the system ening w i l l Other  i s small.  i n t h e v e r y f a s t exchange r e g i o n , l i n e  n o t be v i s i b l e  factors  inhibitors  shift  i f t h e bound i n h i b i t o r  Even broad-  i s flexible.  s u c h a s d e n a t u r a t i o n and a g g r e g a t i o n w h i c h  cause non-broadening  by i n t e r f e r i n g w i t h  might  i n h i b i t o r s binding to  - 80 -  t h e enzyme, a r e r i g o r o u s l y e x c l u d e d as  i n the previous  i s t h e 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 s o y b e a n  inhibitor  present  i n t h e enzyme e x t r a c t .  found not t o a f f e c t the c a t a l y t i c ase,  suggesting  that neither  enzyme b i n d i n g . t h e NMR  trypsin  These p r o t e i n s  activity  were  of a c e t y l c h o l i n e s t e r -  i n t e r f e r s with  the  inhibitor-  The r e a s o n f o r t h e o b s e r v e d n o n - b r o a d e n i n g o f  p e a k s i n t h e p r e s e n c e o f AchE c a n h e n c e be b e s t  p l a i n e d by any o f t h e f o l l o w i n g 1)  chapter,  ex-  cases.  The e x c h a n g e r a t e i s s l o w , w i t h  >> P t , ,  the c o n d i t i o n 2B  being 2)  small chemical species  (Aco) , r e s u l t i n g  be o b s e r v a b l e  c a s e s were d e s c r i b e d  previous  i n no o b s e r v a b l e  in detail  line  line  a  bound  broadening.  broadening  of i n h i b i t o r s  inhibitor  may and  is flexible.  i n S e c t i o n B and C o f t h e  chapter. between  by K a t o and t h e o n e s m e a s u r e d  tract  f a s t , where  t h a t t h e bound  The o b v i o u s d i s c r e p a n c y  explained  u n l i k e l y ) , with  at the c o n c e n t r a t i o n  enzyme u s e d , p r o v i d e d All  (although  s h i f t d i f f e r e n c e between f r e e and  The e x c h a n g e r a t e i s v e r y not  A  met.  The e x c h a n g e r a t e i s f a s t  3)  a  t h e bound l i n e w i d t h s f o u n d  i n t h i s w o r k , c a n be  by t h e d i f f e r e n c e i n p r e p a r a t i o n  o f t h e enzyme e x -  (and h e n c e t h e c o n d i t i o n o f t h e e n z y m e ) .  an enzyme w i t h a s p e c i f i c l i n e hydrolyzed  activity  p e r min p e r mg  p u r i t y of approximately  largely  He  reported  o f 3.3 mmoles o f a c e t y l c h o -  of p r o t e i n , corresponding  to a  3 0 % , a s s u m i n g t h a t s q u i d head AchE i s  - 81  similar sibility  t o e e l enzyme.  -  W i t h s u c h a l o w p u r i t y enzyme, t h e  pos-  o f n o n - s p e c i f i c b i n d i n g has t o be t a k e n 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 linewidths.  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  t h a n e e l enzyme, and  i t i s n o t known w i t h c e r t a i n t y w h e t h e r  aggregative p r o p e r t i e s are s i m i l a r . enzyme u s e d  by K a t o was  t a i n e d no s a l t .  The  aggregated since  c  value calculated  fold  increase  t h e enzyme e x t r a c t  seen  from F i g u r e  i n molecular weight w i l l (  )  thus increase t  by t h e same amount.  and e l e c t r i c  b i n d i n g modes, h e n c e a l t e r i n g —-, the s i m i l a r i t y  activity  among t h e A c h E f r o m d i f f e r e n t  too,  eity,  in inhibitor  specificity sources.  l i n e broadening.  line-  Squid  and  factors  can l e a d  These i n c l u d e magnetic  to  inhomogensuch  ions.  Even when t h e above m e n t i o n e d  s o u r c e s of anomalous  broadening are e l i m i n a t e d , Kato's r e s u l t s are not s i g n i f i c a n t , due  con-  catalytic  i n c r e a s e d v i s c o s i t y , or the p r e s e n c e o f i m p u r i t i e s  as p a r a m a g n e t i c  head  different  Other  b e s i d e s t h e c o n d i t i o n o f t h e enzyme i t s e l f ,  additional  and  c  although i t i s u n l i k e l y  sidering  ten-  Therefore,  by enzyme a g g r e g a t i o n .  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  the  A  i s r e a s o n a b l e t o s u g g e s t t h a t K a t o ' s enormous bound  w i d t h c o u l d be p a r t l y c a u s e d  con-  the  I I - l , and  f r o m t h e assumed m o l e c u l a r w e i g h t .  hence t h e bound l i n e w i d t h it  the  e f f e c t o f t h e s i z e o f t h e enzyme on  bound l i n e w i d t h c a n be r e a d i l y T  I t i s probable that  the  t o t h e n a t u r e o f t h e enzyme.  The  line  especially freeze-dried  - 82 -  A c h E u s e d may n o t h a v e t h e same f o r m a s f r e s h enzyme, s i n c e i t is  known  t h a t a g e i n g h a s a marked e f f e c t on t h e enzyme  activity  (46).  The enzyme was a l s o n o t w e l l c h a r a c t e r i z e d , and most  likely  comprised o f s e v e r a l d i f f e r e n t forms o f AchE.  e i t y o f t h e enzyme of  i s a l s o e s s e n t i a l , s i n c e t h e bound l i n e w i d t h  i n h i b i t o r s d e p e n d s upon t h e s i z e o f t h e enzyme.  only  i f t h e enzyme  to estimate  using  Figure  Kj.  and a l s o g l o b u l a r , w h i c h made i t p o s inhibitor  II-l.  activity  ing of i n h i b i t o r s 80,000 M.W.  i s t h e most s t u d i e d f o r m o f A c h E , w i t h a  identical  t o the n a t i v e  t o t h e 11S t e t r a m e r  catalytic  subunits, with  18S f o r m .  The  mers, they a r e c o n s i d e r e d sites with  respect  The c l a i m t h a t t h i s enzyme to study,  to contain  t h e same b i n d i n g  independent  to i n t e r a c t i o n with species  bind-  i s stoichiometric to i t s constant  As no c o - o p e r a t i v i t y i s o b s e r v e d among t h e s u b u n i t  binding  over-  f o r m o f AchE was u s e d i n t h e  t h e maximum bound l i n e w i d t h f o r an  The 11S s p e c i e s catalytic  To  t h e f o l l o w i n g a d v a n t a g e s ; t h e 11S f o r m i s  s t a b l e , non a g g r e g a t i v e , sible  c a n be e x p e c t e d f o r 7—-.  the l i s  come t h e s e p r o b l e m s , o n l y work, w i t h  Therefore  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  conditions, consistent values  present  Homogen-  mono-  identical  inhibitors.  i s t h e most s u i t a b l e f o r m  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 u s e i n r e c e n t  spectroscopic  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 eserine,  exception) with  the modified  atropine  enzyme p r o d u c e s a s i g n i f i c a n t  b r o a d e n i n g o f t h e NMR r e s o n a n c e s . possible  reasons  that could no  considered  the enzyme-inhibitor  l i n e broadening with  are several interaction) there i s  n a t u r a l enzyme h a s t o be  an e x c e l l e n t c o r r o b o r a t i o n  conditions,  modified  Since there  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  detectable  tal  (besides  and PTA (TMA i s an  of the applied  suggesting the observed  experimen-  l i n e broadening with the  enzyme i s i n d e e d due t o e n z y m e - i n h i b i t o r  interactions.  The  bound l i n e w i d t h s , w h i c h w e r e f o u n d f o r t h e i n t e r a c t i o n o f  the  three  eserine, able  i n h i b i t o r s with  15 Hz f o r PTA, and 98 Hz f o r a t r o p i n e )  and w i t h i n As  t h e s u l f o n y l a t e d enzyme  the previously  described  of the i n h i b i t o r  calculated  i n the previous s e c t i o n  are also  therefore bility  the non-broadening  peaks r e s u l t s e i t h e r from a f a s t or a slow  of the i n h i b i t o r .  of the i n h i b i t o r  flex-  S u l f o n y l a t i o n o f A c h E must  when b o u n d .  L e t us f i r s t  o f a v a r i a t i o n i n t h e exchange  There w i l l  and  a very  have a l t e r e d e i t h e r t h e e x c h a n g e r a t e o r t h e f l e x i -  possibility  if  reason-  l i m i t o f 400 H z .  exchange r a t e , or from a v e r y f a s t exchange w i t h ible binding  (260 Hz f o r  be two a r g u m e n t s u s e d  consider the  rate.  i n order  to investigate  t h e o b s e r v e d s p e c t r a l d i f f e r e n c e between s y s t e m s w i t h modified  First,  enzyme was due t o t h e a l t e r a t i o n o f e x c h a n g e  the p o s s i b i l i t y  natural rate.  o f t h e unmodified system i n t h e slow  - 84  exchange r e g i o n  i s explored.  u n l i k e l y t h a t the Second, i t w i l l t i o n of fast.  the  enzyme c h a n g e s t h e  the  sociation  i n Chapter IV i t i s  f a s t exchange  examined whether  i t is likely  exchange r a t e  d i s s o c i a t i o n rate  r e a c t i o n E + I £ EI ( K j ) by  (As e x p l a i n e d  system i s i n the  be  Here, the  -  (k_  i s r e l a t e d t o the  I f the  that sulfonyla-  from slow to  o f f rate)  l f  dissociation  r e l a t i o n , K^. = k_^/k^, where k^  rate.  region).  for  very  the  constant  represents  the  association rates for inhibitors  asare 9  assumed t o be M  S ,  - 1  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 *  i t i s p o s s i b l e t o c a l c u l a t e the  known d i s s o c i a t i o n c o n s t a n t s . ation constants f o r TMA. for  any  The  slow exchange  very  i t is unlikely  to bind  t o AchE a t  a  inhibitor  t o the by  very  binding  be  the  To  d i s s o c i a t i o n constant  to  natural  change from  off rate  times.  to  i n f e r r e d from  constants  AchE.  fast region,  a p p r o x i m a t e l y 10^  of the  exchange r a t e from slow  enzyme may  those to v a r i o u s modified  increase  the  rate.  s u l f o n y l a t i n g the  must i n c r e a s e an  o f f rates suggest that  i n h i b i t o r s except eserine  s t u d i e s comparing the  slow region  o f f r a t e s from  10  In the p r e s e n t work, the d i s s o c i —6 —3 x 10 M f o r e s e r i n e t o 5 x 10 M  p o s s i b i l i t y of c h a n g i n g the  f a s t by  AchE w i t h  f r o m 3.3  estimated  of the  The  vary  {-  (P  B A  or  the k_-^)  This would r e s u l t i n by  at l e a s t the  same  * T h i s i s a r e a s o n a b l e assumption, c o n s i d e r i n g the f a c t t h a t t h e b i n d i n g o f b o t h 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 ) a p p e a r s t o be d i f f u s i o n c o n t r o l l e d ( 7 7 , 1 1 3 ) .  - 85 -  amount.  According  t o Main  (46) , a c y l a t e d AchE i n d e e d  larger d i s s o c i a t i o n constants ticularly  true f o r s i t e directed i n h i b i t o r s ,  PTA, a l t h o u g h Similar  than n a t u r a l AchE.  the observed  increase  r e s u l t s on t h e b i n d i n g  m y l a t e d AchE were o b t a i n e d bamylation  s u c h a s c h o l i n e and  ligands bearing  and f l u o r e s c e n c e  bulky  measurements  of the d i s s o c i a t i o n constants  ligand  - 3-hydroxyphenyltrimethyl  ( 5 1 ) . The b i n d i n g o f  s u b s t i t u e n t s t o methane-  101),  whereas Suszkiw  pletely abolished. the  binding  spin labels  was f o u n d .  ammonium  an e x c e p t i o n a l d e c r e a s e i n t h e b i n d i n g Wilson  to carba-  ( 7 6 ) . H e r e , a 20 f o l d i n -  crease  s u l f o n y l a t e d AchE.  t h a n 50 f o l d .  inhibitors  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 (79)  i s par-  from s t u d i e s o f the r a t e s o f d e c a r -  i n the presence of i n h i b i t o r s  bisquarternary  This  i s not greater  of various  gives  observed  (102) r e p o r t e d  O n l y one  (3HPTA) - showed  affinity  a 100 f o l d  t o t h e methanei n c r e a s e (92,  the binding  t o be com-  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  o f 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 n o t s i g n i -  f i c a n t l y decrease the binding  affinity  and h e n c e t h e e x c h a n g e  r a t e c a n n o t be a l t e r e d f r o m s l o w t o v e r y  f a s t by m o d i f y i n g  the  enzyme. The preted  value  enzyme, h o w e v e r , must be  c a r e f u l l y and t h e o b s e r v a t i o n  the b i n d i n g binding  f o r modified  constant  constant  of only  inter-  a s m a l l change i n  (K^) d o e s n o t n e c e s s a r i l y mean t h a t t h e  a t the a c t i v e s i t e  c a u s e f o r most l i g a n d s , t h e r e  has h a r d l y changed.  e x i s t s no p r o o f  Be-  that they don't  -  86  -  bind t o p e r i p h e r a l s i t e s as w e l l ; i n f a c t many l i g a n d s , e s p e c i a l l y bisquarternary types o f s i t e .  l i g a n d s , are known t o bind at both  I t may then be p o s s i b l e f o r an i n h i b i t o r  to a p e r i p h e r a l s i t e when an a c t i v e s i t e t h i s case, the b i n d i n g constant  to bind  i s not a v a i l a b l e .  In  f o r the p e r i p h e r a l s i t e , which  c o u l d be q u i t e s i m i l a r t o that f o r an a c t i v e s i t e , would appear to be Kj f o r AchE as a whole, while the a c t u a l change at the o r i g i n a l binding  s i t e might have been much b i g g e r .  f o r 3-HPTA - the only  And indeed  i n h i b i t o r which the non-binding t o p e r i -  p h e r a l s i t e s has been c o n c l u s i v e l y shown f o r to date - a s i g n i f i c a n t change i n modified  has been found f o r the i n t e r a c t i o n with  AchE.  Since  from a l l the arguments d i s c u s s e d  previously,  i t seems  u n l i k e l y that the observed d i f f e r e n c e i n l i n e w i d t h can be a t tributed  to an a l t e r a t i o n o f the exchange rate  methanesulfonylation  following  of the enzyme, w e ' l l now consider the  second p o s s i b l e reason, namely the f l e x i b i l i t y of bound bitors.  I t i s hard t o imagine that a t t a c h i n g  inhi-  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 t o the a n i o n i c s i t e at the a c t i v e s u r f a c e , s i n c e these s i t e s are i n c l o s e p r o x i m i t y . i n h i b i t o r s which bind  t o the a c t i v e a n i o n i c s i t e , a t r o p i n e and  e s e r i n e show a g r e a t e r  value  of =1— compared with PTA.  supports our concept s i n c e a t r o p i n e molecules than PTA.  Of these  This  and e s e r i n e are l a r g e r  The r e s u l t s with TMA and s p i r o  inhibitor  - 87  can  be  explained  m i g h t be  similarly.  the  importance of  provided  by  that  active site  the  the  and/or l o c a t e d  i s a s m a l l m o l e c u l e , and  l i n e b r o a d e n i n g , even w i t h  spiro inhibitor  shows b r o a d e n i n g e v e n w i t h for  TMA  e x p e c t e d , shows no  AchE, whereas the  -  the  as  a very bulky  n a t u r a l enzyme.  following observations.  on  the  region  why,  observed fied.  surface  of  the  enzyme  sulfonyl a t e d by  modification  fluoride,  i t was  of  ( 8 6 , 1 0 3 ) , no  the  the  suggest  s m a l l m o l e c u l e s s u c h as TMA,  the  This  done w i t h  reaction  TeMA and  inhibited  t e t r a - n - b u t y l a m m o n i u m and s u l f o n y l a t i o n (47,68,69).  fonylation  by e s t e r s  those c o n t a i n i n g small  and  b o t h TMA  large  is  TEA  and  where a l a r g e teratic  site.  inhibitor fied  PTA  t o the  inhibitor I t can  can  (101).  anionic  therefore  be  This  site  sterically  linewidth difference  s y s t e m i s due  rather  However t h e  PTA  to  example  rate of s u l particular i s slowed  also  at the  implies  active  interfere with  concluded  between the  exchange r a t e .  t h a t the  modified  t o a change i n f l e x i b i l i t y  than a change i n the  methane-  acceler-  (and  in  a q u a t e r n a r y ammonium f u n c t i o n ,  bind  is  tetra-n-propylammonium,  of methanesulfonic a c i d s ,  inhibitors alike  might  broadening  a much l e s s e r e x t e n t ) , w h e r e a s l a r g e r m o l e c u l e s , f o r gallanine,  large  enzyme i s m o d i -  enzyme was  found t h a t  is  ( 1 0 9 , 1 1 0 ) , i s more  elastase.  present experiment unless  When t h e  studies  of n a t u r a l AchE, which i s  u n l i k e o t h e r enzymes  i n the  evidence  active site  ESR  s p a c i o u s t h a n t h a t o f a - c h y m o t r y p s i n and explain  modified  molecule  Further  geometry around the  as  that  region  the  es-  observed  and  (hence i n (The  by  latter  unmodi7^—)r  - 88  possibility,  however, can  g e s t s , as ESR  not  -  be e n t i r e l y e x c l u d e d . )  This  sug-  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 of n a t u r a l  A c h E i s l a r g e e n o u g h 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 , effective spacious  size being  a t l e a s t the  than t h a t of the  s i z e o f a t r o p i n e , and  ments  (86,112).  The  bound l i n e w i d t h s w i t h  is  bound l i n e w i d t h o f PTA  (  l a r g e enough t o a f f o r d r o t a t i o n a l  m o l e c u l e e v e n when t h e e s t e r a t i c group. C. P O S S I B L E EXTENSION OF  moreover  pharmacologically  drugs.  e n z y m e , h o w e v e r , has  scopic  q u e n t use  has  PTA  w i t h a MeSC^  As m e n t i o n e d e a r l i e r ,  with this  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 e s p e c i a l l y with  spectro-  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 o f h i g h p u r i t y AchE  (by f l u o r e s c e n c e  and  The  ESR)  o n l y been u s e d by one  i s e x p e c t e d f o r the  able of monitoring  enzyme  for i t s interaction  the  recent  provided  a b o u t t h i s enzyme a t t h e m o l e c u l a r  spectroscopy  the  interest for i t s e f f i c i e n t  been a v a i l a b l e u n t i l r e c e n t l y .  s t u d i e s on A c h E formation  i s blocked  related serine proteases,  techniques,  have n o t  not  of  STUDIES  l o n g been o f r e s e a r c h  important  to  2B  f r e e d o m o f a bound  site  m e c h a n i s m o f h y d r o l y s i s and  the c l o s e l y  = 15 Hz)  1  AchE a l s o i n d i c a t e s t h e a c t i v e c e n t e r  A c h E has  inhibi-  t o t h e o n e s u s e d i n the c u r r e n t e x p e r i 1 T A  the m o d i f i e d  more  r e l a t e d enzyme, a - c h y m o t r y p s i n , w h i c h  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 t o r s comparable i n s i z e  the  spectroscopic detailed in-  l e v e l . Although  g r o u p so f a r , more  future, since this  i n t e r a c t i o n o f any  NMR  fre-  technique  l i g a n d w i t h AchE.  i s capAs  - 89  larger  -  amounts o f p u r e AchE a r e now  becoming a v a i l a b l e , the  signal-to-noise  r a t i o o f NMR  techniques  become l e s s i m p o r t a n t .  will  also achieved  by m o n i t o r i n g  most i n h i b i t o r s  and  drugs.  sociation constants  compared t o the  a CH^  Greater  spectroscopic  sensitivity  is  group, which i s contained  in  Choosing  (K^.) w i l l  other  inhibitors  with small  concluded  inhibitors,  as  t h a t the  smaller  the m o l e c u l e s w i t h  undergo exchange a t a slower  the  a l s o be  and  homogeneous m a g n e t i c f i e l d s  and  with high  The  l a r g e l y o v e r c o m e by u s i n g NMR  t o use  However i t  small concentrations  drawback of  r a t i o o f bound t o f r e e i n h i b i t o r  D  with  high  magnets)  make i t p o s -  inhibitors  (f ).  to  insensitivity  spectrometers  This w i l l  of  the  a r e more l i k e l y  (e.g. s u p e r c o n d u c t i n g  signal-to-noise ratio.  by  t h e more u s e f u l  smaller  rate.  can  sible  dis-  also give further s e n s i t i v i t y  a l a r g e r c o n t r i b u t i o n o f s i g n a l s o f bound i n h i b i t o r . c a n n o t be  poor  giving better  From t h e e q u a t i o n s  in  s l o w and  ex-  hi Table I I - l i t i s c l e a r t h a t i n both the change r e g i o n will to  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  increase with  l a r g e r f„.  H e n c e , i t may  o b s e r v e a b r o a d e n i n g o f t h e NMR  inhibitors  with  Once any  l i n e broadening  interaction  of  inhibitor  con-  i s observed with  MHz natural  be d e t e r m i n e d , r e s o l v i n g t h e  discussed  e a r l i e r , as t o w h e t h e r t h e  tion  c h a n g e d e i t h e r t h e e x c h a n g e r a t e s or inhibitors.  possible  t h e c a s e w i t h a 270  question  o f bound  fast  broadening  w e l l be  peaks i n the  u s e d , as w o u l d be  enzyme, t h e e x c h a n g e r e g i o n c a n  has  line  t h e n a t u r a l enzyme, i f a s m a l l e r  c e n t r a t i o n c o u l d be spectrometer.  very  methanesulfonylathe  flexibility  - 90 -  Also,  i f t h e bound l i n e w i d t h o f an i n h i b i t o r  w i t h t h e n a t u r a l enzyme c o u l d be o b t a i n e d , NMR  interacting  spectroscopy  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 f o r m a t i o n a l changes i n AchE. which suggested  such  b i n d i n g t o AchE.  T h e r e have been many  a change b e i n g  experiments  associated with a ligand  For instance, i t i s widely accepted  influence of quaternary  con-  t h a t the  i n h i b i t o r s on t h e enzyme a c t i v i t y i s  b r o u g h t a b o u t 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 a n d a c o n c o mitant  c o n f o r m a t i o n a l change o f AchE  c l u s i o n s h a v e been r e a c h e d zyme by i r r e v e r s i b l e of phosphoric inhibitors  inhibitors,  such  a c i d s i n the presence  Bolger  a s h i f t o f t h e e m i s s i o n maximum o f p r o t e i n  conclusive evidence AchE i s g i v e n study  site  Most o f  t o provide d i r e c t evidence.  The  first  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 o f  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 ( 1 0 5 ) . I n with the  a t t h e a c t i v e - c e n t e r a r e employed t o demonstrate  that peripheral site directed ligands alter  conformation.  sensitive  (66).  f l u o r e s c e n t phosphonates which conjugate  s e r i n e moiety directly  w i t h A c h E and a s l o w u n i -  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 o f  t h e enzyme, h o w e v e r , f a i l  their  fluorescence study i n  i n t e r c o n v e r s i o n o f t h e enzyme s p e c i e s  the e x p e r i m e n t a l  of reversible  and T a y l o r a l s o p r o p o s e d a c o n f o r -  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 molecular  con-  a s c a r b a m a t e s and e s t e r s  m a t i o n a l c h a n g e o f t h e enzyme f r o m t h e i r which they observed  Similar  by s t u d y i n g t h e a c y l a t i o n o f t h e e n -  and s u l f o n i c  (47,51).  (21,69,104).  the a c t i v e  The NMR l i n e w i d t h o f a bound i n h i b i t o r  t o the surrounding  i s very  e n v i r o n m e n t and i t a l s o s h o u l d  -  provide information near the binding 1.  -  91  about a c o n f o r m a t i o n a l change of the enzyme  site.  Such an experiment can be designed by  modifying the p e r i p h e r a l  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 l i g a n d a s s o c i a t i n g  with  the a c t i v e s i t e , or 2.  l e t t i n g a l i g a n d , which a s s o c i a t e s center,  only with the a c t i v e  i n t e r a c t with AchE i n the presence and absence of  a peripheral  site directed  l i g a n d and comparing the value  ~ — values of the a c t i v e s i t e d i r e c t e d 2B  ligand.  1  As mentioned i n Chapter I I , binding are u n f o r t u n a t e l y very l i t t l e  not c l e a r l y e l u c i d a t e d  binding  experiments i s thus r e s t r i c t e d to compounds such as  peripheral  sites, respectively.  of the enzyme can a l s o be s t u d i e d  e x c l u s i v e l y t o the a c t i v e The c o n f o r m a t i o n a l change  by modifying the a c t i v e  of the enzyme with a c i d t r a n s f e r i n g An  since  The c h o i c e of i n h i b i t o r s f o r the above  edrophonium and propidium, which bind and  yet, especially  i s known so f a r about the extent o f t h e i r  to secondary s i t e s . described  modes f o r most i n h i b i t o r s  site  irreversible inhibitors.  example of the i n v e s t i g a t i o n of the a c t i v e s i t e area with  a NMR  technique using  an a c i d t r a n s f e r i n g  i n h i b i t o r i s that of  that by Reech e t a l , i n which d i i s o p r o p y l f l u o r o p h o s p h a t e vatives  deri-  of chymotrypsin and chymotrypsinogen are compared by  phosphorus-31  NMR  (111).  of i n h i b i t o r s c o v a l e n t l y  In our case, d i r e c t  observation  bound to the e s t e r a t i c s i t e with  spectroscopy i n the presence and absence of other l i g a n d s elucidate  the p o s s i b l e  a l l o s t e r i c e f f e c t of l i g a n d s  NMR might  b i n d i n g to  - 92  the p e r i p h e r a l s i t e . here i s t h a t the unlike  the  t e n t of  The  advantage of using  change i n the  information  c o n c e r n i n g the TJT — 1  i  hibitor  molecule.  This  of the  required  i n answering a long  the  i n f l u e n c e of  ligands accelerate  reaction while binding  separately  the  s i t e s near the  inhibitor,  a measure of  other  the  ex-  It is possible  to gain  nature of  conformational  for various  the  regions  more  o f an i n -  2B  ligand binding  substrate decelerate.  under-  on A c h E w i l l  sought q u e s t i o n :  why  be  certain  peri-  (or i r r e v e r s i b l e i n h i b i t o r ) The  ability  a t d i f f e r e n t groups w i t h i n the will  spectroscopy  type of p r e c i s e m o l e c u l a r l e v e l  standing  pheral  provides  change.  change o f A c h E by d e t e r m i n i n g  NMR  l i n e w i d t h o f a bound  change i n f l u o r e s c e n c e ,  the c o n f o r m a t i o n a l  detailed  -  to monitor  inhibitor  molecule  assist in identifying possible multiple a c t i v e r e g i o n , p r o p o s e d by  two  workers  binding  (46,107),  and c h a r a c t e r i z i n g them. F o r i n s t a n c e , t h e r e s p o n s e o f t h e l i n e w i d t h (-^-) o f b o t h a t r o p i n e p h e n y l and N - m e t h y l p e a k s (and 2 i  a l s o of pine  t r o p i n e and  molecule, containing  phenyl ring) could p.H.,  t r o p i c a c i d , the  be  two  subunits  of  the  e i t h e r c h a r g e d m e t h y l g r o u p or  studied  under the  atrothe  i n f l u e n c e of d i f f e r e n t  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 m e a s u r e i n t h e a n a l y s i s 2B A  inhibitor  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  obtainable region. provides in  o n l y when t h e  A study of b a s i c a l l y the  Table I I - l ) ,  system i s i n the  very  level,  of  i t is  f a s t exchange  under the exchange c o n d i t i o n s , which 1 same i n f o r m a t i o n as t h a t o f 7 = 7 — (so shown 2 i s much l e s s f r e q u e n t l y p e r f o r m e d s i n c e t h e 1  l  - 93 -  measurements are more time consuming.  =^- however, can be f  1 i s not f a s t enough and = — cannot 2B 1  u s e f u l when the exchange rate  i  be o b t a i n e d .  The exchange rate  in T^ time s c a l e than i n T  2  i s more l i k e l y t o be very f a s t  time s c a l e because  i s indepen-  dent of Ato (as seen i n Table I I - l ) and T^ i s g e n e r a l l y  larger  than T . Hence i t i s s t i l l p o s s i b l e to c a l c u l a t e 7 =^—,. which ^ lB p r o v i d e s the same type of i n f o r m a t i o n as — — , while the system , 2B i s too slow t o c a l c u l a t e 7=— and such a case i s indeed r e p o r t e d 2B by Sykes (103). 0  i  X  l  - 94 -  GLOSSARY OF TERMS AND ABBREVIATIONS  Ach: AchE:  acetylcholine acetylcholinesterase  allosteric  site:  a binding can b i n d the  ESR:  electron  esteratic  spin  site:  site  on an enzyme where a l i g a n d  and cause a s t r u c t u r a l change i n  enzyme m o l e c u l e  resonance  a site  c o n s i s t i n g of the residues  which  d i r e c t l y p a r t i c i p a t e i n t h e m a k i n g and breaking HPTA: NMR:  3-hydroxyphenyltrimethylammonium i o n nuclear  proteolysis: PTA: serine  o f bonds  magnetic  resonance  enzymic h y d r o l y s i s o f peptide  phenyltrimethylammonium i o n protease:  an enzyme w h i c h c a n s p l i t c e r t a i n linkages  a t any p o i n t  i n a peptide  and have a r e a c t i v e s e r i n e TeMA: TMA:  bonds  tetramethylammonium i o n trimethylammonium i o n  residue  peptide chain  -  95  -  REFERENCES  1.  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