Theoretical Modeling of Enzymic Hydrolysis of Acetylcholine

14 Theoretical Modeling of Enzymic Hydrolysis of Acetylcholine Compared to Acetylthiocholine JOYCE H . CORRINGTON

Downloaded by COLUMBIA UNIV on November 29, 2017 | http://pubs.acs.org Publication Date: November 28, 1979 | doi: 10.1021/bk-1979-0112.ch014

Xavier University of Louisiana, New Orleans, L A 70125

Acetylcholine (ACh, 51-84-3) is a molecular ion which functions as a neurohumoral transmitter in the peripheral nervous system. It is released at the end of one cell, diffuses across the synapse, induces permeability changes in the next cell, and is then rapidly removed from the synaptic region by a hydrolytic reaction catalyzed by the enzyme acetylcholinesterase (AChE, EC 3 . 1 . 1 . 7 ) . Replacement of the acetate oxygen of ACh by sulfur y i e l d i n g the thiol ester analog a c e t y l t h i o choline (ASCh, 4468-05-7) profoundly modifies the b i o l o g i c a l action of this molecular ion ( 1 , 2 , 3 ) . The r e l a t i v e a b i l i t y of these isosteres to induce p o l a r i z a tion in isolated single c e l l electroplax preparations is 1.0:16.7 (4). However, the r e l a t i v e rate at which these isosteres are hydrolyzed by AChE is very s i m i l a r , 1.0:0.6 (5). Proposals made concerning this hydrolysis mechanism suggest that the active s i t e of AChE consists of a nucleophilic serine residue and a h i s t i d i n e residue which probably serves as a proton donor or receiver (6). Hydrolysis is thought to occur through the formation of a Michealis complex between the active s i t e of the AChE and the substrate (S), the conversion of the complex in an "acylation" reaction to an acyl enzyme intermediate (AChE•A) and the product choline (or t h i o c h o l i n e ) , and the subsequent hydrolysis of the acyl enzyme in a "deacylation" reaction to acetic acid and the regenerated enzyme ( 7 , 8 , 9 ) .

The a c y l a t i o n

reaction

appears

controlled

by a f u n c t i o n a l

0-8412-0521-3/79/47-112-301$05.00/0 © 1979 American Chemical Society

Olson and Christoffersen; Computer-Assisted Drug Design ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


302

COMPUTER-ASSISTED DRUG DESIGN

g r o u p o f p K = 5.3, w h i l e t h e d e a c y l a t i o n r e a c t i o n a p p e a r s c a t a l y z e d b y a g r o u p o f p K = 6.3, a n d f o r b o t h A C h a n d ASCh t h er a t e d e t e r m i n i n g s t e p i s t h ed e a c y l a t i o n reac t i o n (10J. Semiempirical m o l e c u l a r o r b i t a l c a l c u l a t i o n s have b e e n e m p l o y e d t o c o m p a r e t h e c o n f o r m a t i o n [11) a n d t h e e l e c t r o n i c p r o p e r t i e s o f A C h a n d A S C h (1_2). T w o s t u d i e s have i n v e s t i g a t e d proposed h y d r o l y s i s mechanisms f o r ACh (1_3,1_4). T h i s s t u d y s i m i l a r l y e m p l o y s semiempirical c a l c u l a t i o n s t o i n v e s t i g a t e t h ecommonly p r o p o s e d steps o f the mechanism f o r the AChE c a t a l y z e d h y d r o l y s i s o f ACh a n d A S C h . In t h i s s t u d y t h e m o l e c u l a r o r b i t a l c a l c u l a t i o n p r o g r a m e m p l o y e d , ARCANA, u t i l i z e d o n l y a t o m i c d a t a a n d i t e r a t e d t o charge s e l f - c o n s i s t e n c y (see Cal c u l a t i o n Note). T h e s u b s t r a t e s , ACh a n d ASCh, and t h e products of hydrolysis, choline, thiocholine, and acetic acid, were r e p r e s e n t e d i n t h e i r e n t i r e t y , w h i l e the AChE e n z y m i c a c t i v e s i t e was m o d e l e d b yr e p r e s e n t i n g t h e h i s t i d i n e r e s i d u e a n d t h es e r i n e residue thought t o be at t h ea c t i v e s i t e b yimidazol a n d methanol r e s p e c t i v e l y . M o l e c u l a r o r b i t a l c a l c u l a t i o n s have been p e r f o r m e d f o r reactants, three possible intermediate a c y l a t i o n com p l e x e s , and t h ea c y l a t i o n products. Since the deacyla t i o n mechanism i s i d e n t i c a l f o r both ACh a n d ASCh a n d has been r e p o r t e d e l s e w h e r e ( 1 4 ) , t h o s e r e s u l t s w i l l not ber e p e a t e d here. F o r eacTT o f t h e a c y l a t i o n steps, three cases were c a l c u l a t e d : ( a ) general acid catalyzed, (b) n o n - c a t a l y z e d o r n e u t r a l , a n d (c) general base c a t a lyzed. Molecular orbital coefficients and energies, t o t a l valence e n e r g i e s , Mulliken n e tatomic charges, bond a n d t o t a l o v e r l a p p o p u l a t i o n s , a n d bond a n d t o t a l o v e r l a p energies were c a l c u l a t e d f o r each run. The a t o m i c c o o r d i n a t e s used f o r ACh were those e m p l o y e d i n a p r e v i o u s t h e o r e t i c a l s t u d y (15J a n d w e r e calculated employing i d e a l i z e d hybridization and average bond d i s t a n c e s . While c r y s t a l l o g r a p h i c s t u d i e s o f ACh are a v a i l a b l e ( 1£,Γ7), the gauche c r y s t a l c o n f o r m a t i o n o f ACh was n o tused f o r t h i s s t u d y , b u t r a t h e r an a n t i planar conformation s i m i l a r t o t h et r a n s i t i o n state g e o m e t r y s u g g e s t e d b y k i n e t i c d a t a (TJB-22J . T h e a t o m i c coordinates f o r ASCh i n i t s a n t i - p l a n a r c r y s t a l s t r u c t u r e w e r e e m p l o y e d (23). T h e ASCh c r y s t a l l o g r a p h i c bond d i s t a n c e s a n d the ACh g e n e r a l i z e d bond d i s t a n c e s were i d e n t i c a l w i t h i n 0.04 A , t h e a c c u r a c y o f t h e c r y s t a l l o graphic data, with t h e exception o f those involving sulfur. T a b l e I presents t h ebond d i s t a n c e s employed in these c a l c u l a t i o n s f o r t h e s u b s t r a t e , e i t h e r ACh o r ASCh.


14. coRRiNGTON

Enzymic Hydrolysis of Acetylcholine

303

Table I Interatomic

Distances

(8)


X =

X =

1.42

1.97

1.31

1.77

1.23

1.23

1.52

1.52

0.96

1.33

1.09

1.09

1.48

1.48

The i m i d a z o l c o o r d i n a t e s were t a k e n from a n x - r a y d i f f r a c t i o n c r y s t a l l o g r a p h i c study of histamine (24) with a proton being s u b s t i t u t e d f o r the ethylamine s i d e chain of histamine. The c o o r d i n a t e s o f o t h e r r e a c t a n t s , p r o d u c t s , and i n t e r m e d i a t e c o m p l e x e s were c a l c u l a t e d e m p l o y i n g i d e a l i z e d h y b r i d i z a t i o n and a v e r a g e bond distances o rare as d i s c u s s e d . Results the

T h e a c y l a t i o n m e c h a n i s m was four steps following:

assumed

t oc o n s i s to f

Step I. The r e a c t a n t s (ACh o r A S C h , m e t h a n o l , i m i d a z o l ( s ) and/or i m i d a z o l i u m ( s ) form a " p l a n a r " i n t e r mediate complex between the carbonyl carbon of the s u b s t r a t e (which i s in an s p " p l a n a r " h y b r i d i z a t i o n s t a t e ) and t h e o x y g e n o f t h e a t t a c k i n g n u c l e o p h i l e methanol. The n u c l e o p h i l e i s assumed t o a t t a c k p e r p e n d i c u l a r to the plane of the s u b s t r a t e a c e t a t e (or t h i o a c e t a t e ) moity with the e l e c t r o n s from the methanol oxygen f i l l i n g the lowest empty m o l e c u l a r o r b i t a l which is centered on the carbonyl carbon of the s u b s t r a t e . Step moity of with the zatioa of to s p .

I I . The " p l a n a r " a c e t a t e ( o r t h i o a c e t a t e t h e ACh ( o r ASCh) f o r m s a " t e t r a h e d r a l " c o m p l e x a t t a c k i n g n u c l e o p h i l e methanol as the h y b r i d i the s u b s t r a t e carbonyl carbon changes from s p

Step I I I . A "protonated formed by the t r a n s f e r of the

ester" intermediate hydroxy proton from


is the

2

304


a t t a c k i n g n u c l e o p h i l e methanol t o the acetate oxygen (or t h i o a c e t a t e s u l f u r ) , except i n the base c a t a l y z e d pathway where t h i s p r o t o n h a s a l r e a d y been donated t o an i m i d a z o l , a n d t h i s s t e p i s n o t a s s u m e d .


Step IV. The "protonated ester" intermediate breaks i n t o c h o l i n e (or t h i o c h o l i n e ) a n d an a c y l enzyme i n t e r m e d i a t e , modeled here by methyl acetate. Each o f the above s t e p s was c a l c u l a t e d f o r t h r e e pathways: (a) assuming an i m i d a z o l i u m p r e s e n t a t the AChE a c t i v e s i t e a c t e d a s a g e n e r a l a c i d c a t a l y s t a n d donated a proton t o the a c y l oxygen o f the s u b s t r a t e (thus two i m i d a z o l s were i n c l u d e d i n the c a l c u l a t i o n s ) , (b) a s s u m i n g t h e r e was no a c i d o r base c a t a l y s t ( t h u s one i m i d a z o l a n d o n e i m i d a z o l i u m were i n c l u d e d i n t h e c a l c u l a t i o n s ) , a n d (c) assuming an i m i d a z o l present at the AChE a c t i v e s i t e a c t e d a s a g e n e r a l base c a t a l y s t and a b s t r a c t e d a p r o t o n from t h e n u c l e o p h i l e m a k i n g i t a methoxy (thus two imidazoliums were i n c l u d e d i n t h e calculations). I n a l l cases the number o f atoms p r e s e n t were thus kept c o n s t a n t from run t o run. S i n c e no c r y s t a l l o g r a p h i c data o f the a c t i v e s i t e are a v a i l a b l e , modeling o f the s p e c i f i c i n t e r a c t i o n o f the h i s t i d i n e residue with the proposed r e a c t i o n intermediate complexes was n o t a t t e m p t e d . While a f e w angles a n d d i s t a n c e s were v a r i e d d u r i n g the modeling, i ngeneral these were not optimized. T h ed i s t a n c e between the s u b s t r a t e carbonyl carbon andthe n u c l e o p h i l i c oxygen i nthe " p l a n a r " i n t e r m e d i a t e complex was s e l e c t e d so t h a t the c o o r d i n a t e s of the n u c l e o p h i l e d i d not have t o be a l t e r e d as t h e s u b s t r a t e changed h y b r i d i z a t i o n and formed the " t e t r a h e d r a l " i n t e r m e d i a t e complex with a normal carbon t o oxygen bond d i s t a n c e . Discussion Total Valence Energy. Thecalculated total valence e n e r g i e s presented below i nTable I I are the e q u i v a l e n t of t o t a l e n e r g i e s but i n c l u d e o n l y f a c t o r s d u e t o the valence e l e c t r o n s and the nuclear charges corresponding to them. A l l runs were converged t o a 1 kcal/mole t o l e r a n c e , but s i n c e a r b i t r a r y a n d r i g i d conformations were employed these e n e r g i e s should be taken o n l y as i n d i c a t i v e o f the energy changes i n v o l v e d . A l s o , o f course, entropy and s o l v a t i o n e f f e c t s are not included i n such i s o l a t e d o r "gas p h a s e " m o l e c u l a r c a l c u l a t i o n s . But even w i t h t h e s e q u a l i f i c a t i o n s , the t o t a l v a l e n c e e n e r g i e s i n d i c a t e t h a t both ACh a n d ASCh would f o l l o w a l o w e r e n e r g y a c y l a t i o n p a t h w a y when a c i d c a t a l y z e d (when




14. coRRiNGTON

305

Table I I Total Valence Energy f o r Steps i n the Enzymic A c y l a t i o n o f ACh and ASCh ( g - k c a l / m o l e , r e l a t i v e t o t h e r e a c t a n t s ) M e c h a n i sm Step

(A) A c i d Catalyzed

(B) NonCatalyzed

(C) Base Catalyzed

S u b s t r a t e : ACh

ASCh

ACh

ASCh

ACh

ASCh

I II III IV

25 15 -58

162 96 138 57

153 110 120 79

315 283

293 274

-12 -63 -47

-



306


the i m i d a z o l i u m assumed present a t the c a t a l y s t ' s a c t i v e s i t e donates a proton t o the acyl oxygen o f the substrate). T h e energies o f the ACh "planar" and" t e t r a h e d r a l " i n t e r m e d i a t e complexes are lower than those f o r ASCh. Both the A C h a n d the ASCh " t e t r a h e d r a l " i n t e r m e d i a t e s are lower i nenergy than the corresponding "planar" i n t e r m e d i a t e s , as might be expected. A notica b l e d i f f e r e n c e e x i s t s i n t h e i n t e r m e d i a t e f o r m e d when a proton i s s h i f t e d from the a t t a c k i n g n u c l e o p h i l e t o the acetate oxygen (or t h i o a c e t a t e s u l f u r ) , s i n c e the ACh i n t e r m e d i a t e i s n o t a s s t a b l e a s i t s " t e t r a h e d r a l " i n t e r m e d i a t e , w h i l e the ASCh i n t e r m e d i a t e i s c o n s i d e r a b l y more s t a b l e than i t s " t e t r a h e d r a l " i n t e r m e d i a t e a n d indeed roughly e q u i v a l e n t t o the ACh " t e t r a h e d r a l " intermediate. These ACh a n d ASCh i n t e r m e d i a t e s are both more s t a b l e than the proposed a c y l a t i o n p r o d u c t s c h o l i n e (or t h i o c h o l i n e ) a n d the a c y l enzyme (modeled here by methyl a c e t a t e ) . In both cases the a c y l a t i o n r e a c t i o n is c a l c u l a t e d t o be endothermic. S i n c e the r e s u l t i n g a c y l enzyme i s i d e n t i c a l f o r both s u b s t r a t e s , the d e a c y l a t i o n r e a c t i o n would be i d e n t i c a l f o r both and i s not d i s c u s s e d here. However, a s i m i l a r p r e v i o u s s t u d y (1_4) d i d i n d i c a t e t h a t d e a c y l a t i o n would have t o procedle t h r o u g h a h i g h e r e n e r g y pathway than e i t h e r the A C h o r the ASCh a c y l a t i o n , a n d thus would be r a t e l i m i t i n g as experimental data suggests (10). M u l l i k e n Net A t o m i c C h a r g e s . Thenet atomic charges calculated according t o a Mulliken population analysis (25) are p r e s e n t e d below i nT a b l e I I I f o r the l o w e n e r g y or general a c i d c a t a l y z e d pathway. Both ACh a n d ASCh are molecular ions with p o s i t i v e l y charged c a t i o n i c "heads." T h e c a l c u l a t e d t o t a l M u l l i k e n net atomic c h a r g e s on the onium m e t h y l s o f both ACh a n d ASCh t o t a l approximately 0.98and s t a y e s s e n t a i l l y constant throughout the proposed mechanism steps. S i m i l a r l y , the net charge on the onium n i t r o g e n i si n i t i a l l y a negative -0.31 ( i n c o n t r a s t t o common n o t a t i o n w h i c h d e n o t e s t h e nitrogen as p o s i t i v e l y charged). The n i t r o g e n net charge becomes somewhat l e s s n e g a t i v e i n the proposed mechanism s t e p s , but net charges i nthe t o t a l onium f u n c t i o n a l g r o u p s i nACh, ASCh, c h o l i n e a n d t h i o c h o l i n e s t a y essentailly constant and equivalent. The c a r b o n y l o x y g e n i s t h e most n e g a t i v e atom i n both s u b s t r a t e s , a n d h a s a M u l l i k e n net charge o f -0.698 in ACh a n d -0.661 i nASCh. Centered on i t i nboth subs t r a t e s i s the highest occupied molecular o r b i t a l , thus the c a r b o n y l oxygen i s the most s u b j e c t t o e l e c t r o p h i 1 i c attack or protonation. In the intermediated formed i n



I V

τ TA ιπ ι

2

0.,364 0.,396 0,.447 0..439 0,. 3 6 3

0,,306 0,.361 0,. 4 3 4 0..395 0,.410

- 0 .,670 - 0 ..492 - 0 ,,348 - 0 ..625 -0,. 6 6 2

0..877 0,,958 0,.932 0,.907 0 .895

. „ ^ c (

-0. -0. -0. -0. -0.

-

698 5 2 9 0,.376 5 9 3 0,.341 5 9 3 0,.357 712

+

0.100

0. 101 Q. 1 3 6 0. 0 7 4 0. 0 9 0 0. 0 6 9

3

===== 0 - " H ) - ( C H )

0.118 0.364 0.306 -Q.670 0.438 -0.661

- 0 .,612 - 0 ..558 - 0 , .606 - 0 , .379 - 0 .. 6 1 4

- X

I n t e r m e d i a t e s f o r S u b s t r a t e s ACh and ASCh

0.962 -0.268 0.308 0.067 0.429 0.469 - 0 . 3 6 6 0.551 -0.58Q 0.351 0.077 0 979 -0.251 0.394 0 . 5 4 2 0.1 51 0 . 3 7 0 - 0 . 6 4 6 0 . 5 9 7 - 0 . 5 9 1 0 . 3 6 8 0 . 0 8 8 (K972 -0.310 Q.242 -0.021 0.067 0.410 -0.662 0.895 -0.712 0.069

0.976 -0.308 0.334

Methanol

0,,669 0..644 0,.625 0..679 0,. 5 9 4

2

-(CH )

ASCh

- 0 .,314 - 0 .,266 - 0 .,269 - 0 ..252 - 0 ,.316

+

for Acylation

0..986 0..966 0.. 9 6 4 0..981 0,.972

- N

Charges

Reactants ACh, Methanol IA 11A 111A IV

3

(H C)

Mechanism Step

3

Net Atomic

Mulliken

Table III



308


the a c i d c a t a l y z e d pathway, t h i s oxygen i sassumed t o be protonated by a proton donated by an imidazolium a t the AChE's a c t i v e s i t e . The M u l l i k e n net charge o f t h i s o x y g e n becomes s l i g h t l y l e s s n e g a t i v e when p r o t o n a t e d , but i s s i m i l a r whether the s u b s t r a t e i s ACh o r ASCh. The p r o t o n r e t a i n s o n l y a f r a c t i o n o f i t s n e t p o s i t i v e c h a r g e , v a r y i n g f r o m 0.341 t o 0.382 among t h e proposed intermediates. I t thus apparently withdraws c o n s i d e r a b l e e l e c t r o n d e n s i t y , but not from the a d j a c e n t a c y l oxygen, r a t h e r from the oxygen o f the a t t a c k i n g n u c l e o p h i l e , thus h e l p i n g s t a b i l i z e the d e v e l o p i n g bond between the s u b s t r a t e and the a t t a c k i n g n u c l e o p h i l e . The c a r b o n y l c a r b o n i s t h e most p o s i t i v e atom i n both s u b s t r a t e s , but i sc o n s i d e r a b l y more p o s i t i v e i n the ACh (0.877) than i n the ASCh (0.438). However,t h e lowest energy empty m o l e c u l a r o r b i t a l (LEM0), which i s c e n t e r e d on t h i s atom p e r p e n d i c u l a r t o the plane o f the acetate (or t h i o a c e t a t e ) moity, hass i m i l a r o r b i t a l e n e r g i e s f o r both the A C h (-12.00 e V ) a n d the ASCh (-12.35 e V ) . Both the p o s i t i v e M u l l i k e n net atomic c h a r g e a n d t h e l o c a t i o n o f t h e LEMO o n t h e c a r b o n y l c a r b o n make i t s u b j e c t t o n u c l e o p h i l i c a t t a c k . In the i n t e r m e d i a t e s formed i n the proposed a c i d c a t a l y z e d mechanism s t e p s , the M u l l i k e n net atomic charges o f the carbonyl carbon does not change g r e a t l y d e s p i t e the f o r m a t i o n o f the d e v e l o p i n g bond between the s u b s t r a t e and the a t t a c k i n g n u c l e o p h i l e . The a c e t a t e o x y g e n i st h e s e c o n d s i t e o f n e t n e g a t i v e charge i n the ACh s u b s t r a t e (-0.612), but the corresponding t h i o a c e t a t e s u l f u r hasa net p o s i t i v e charge i n the ASCh s u b s t r a t e (+0.118). This difference i n net c h a r g e h a s been p r o p o s e d a s t h e e x p l a n a t i o n why the c r y s t a l s t r u c t u r e o f ACh a n d ASCh a r e d i f f e r e n t ( 1 2 ) . I t may a l s o e x p l a i n why the t o t a l v a l e n c e e n e r g i e s o f the proposed ASCh " p l a n a r " a n d " t e t r a h e d r a l " a c y l a t i o n i n t e r m e d i a t e s are higher than the corresponding ACh intermediates. T h i s a c e t a t e oxygen (and t h i o a c e t a t e s u l f u r ) i s a l s o the primary s i t e o f the second h i g h e s t energy o c c u p i e d m o l e c u l a r o r b i t a l , an a n t i - b o n d i n g pi-type o r b i t a l shared with the carbonyl oxygen. Because o f t h i s , i t was proposed t h a t a p o s s i b l e a c y l a t i o n i n t e r m e d i a t e (111A) c o u l d r e s u l t i f a proton was t r a n s f e r r e d from the hydroxy o f the a t t a c k i n g n u c l e o p h i l e t o the acetate oxygen (or t h i o a c e t a t e s u l f u r ) . This intermediate r e s u l t e d i n a moderate p o s i t i v e change i nthe net atomic charge o f the a c e t a t e oxygen, but a l a r g e p o s i t i v e change i nthe net charge o f the t h i o a c e t a t e s u l f u r . In t h e s e p a r a t e d c h o l i n e m o l e c u l a r i o n , t h e h y d r o x y oxygen r e t u r n s t o a net atomic charge (-0.614) s i m i l a r


14.

coRRiNGTON


309

t o w h a t i t was i n t h e c h o l i n e m o i t y o f t h e A C h , while in the separated t h i o c h o l i n e m o l e c u l a r ion the mercaptan s u l f u r has a more n e g a t i v e net a t o m i c c h a r g e (-0.021) than i t had i n t h e t h i o c h o l i n e m o i t y o f ASCh.


Bond O v e r l a p P o p u l a t i o n . The c a l c u l a t e d bond o v e r l a p p o p u l a t i o n s f o r some o f t h e b o n d s i n v o l v e d i n the a c y l a t i o n r e a c t i o n are presented below i n Table IV. The o v e r l a p p o p u l a t i o n (OP) b e t w e e n a t o m s A and Β i s c a l c u l a t e d by:

w h e r e i and j a r e summed o v e r a l l a t o m i c b a s i s o r b i t a l s and u o v e r a l l m o l e c u l a r orbitals. I n t h e A C h s u b s t r a t e , t h e OP i n d i c a t e s a h i g h e r e l e c t r o n d e n s i t y (0.698) between the carbonyl carbon and t h e a c e t a t e o x y g e n t h a n the e l e c t r o n d e n s i t y ( 0 . 5 7 9 ) b e t w e e n t h e a l k y l c a r b o n and t h e a c e t a t e o x y g e n due t o p a r t i a l pi-bonding between the a c e t a t e oxygens. However, the ASCh c o r r e s p o n d i n g e l e c t r o n d e n s i t y (0.980) between the c a r b o n y l c a r b o n and the t h i o a c e t a t e s u l f u r i s e v e n l a r g e r due t o e v e n m o r e p a r t i a l p i - b o n d i n g b e t w e e n t h e t h i o a c e t a t e s u l f u r and t h e c a r b o n y l o x y g e n . This enhanced pi-bonding occurs without expanding the atomic b a s i s s e t f o r t h e s u l f u r a t o m t o i n c l u d e e x t r a v a l e n t d_ o r b i t a l s ( 1_2). Since this ester (thioester) linkage i s t h e one w h i c h c o m m o n l y f i s s i o n s i n e s t e r h y d r o l y s i s , i t i s obvious t h a t t h i s r e l a t i v e l y s t r o n g bond must weaken b e f o r e the c h o l i n e ( t h i o c h o l i n e ) m o i t y can depart to conclude the a c y l a t i o n r e a c t i o n . It is observed that the formation of the proposed " t e t r a h e d r a l " intermediate d i s t u r b e s t h i s p a r t i a l pi-bonding such that the e l e c t r o n d e n s i t y o f the e s t e r l i n k a g e f a l l s t o 0.637 (and the t h i o e s t e r l i n k a g e to 0.734), but not to a l o w e r e l e c t r o n d e n s i t y than that of the adjacent a l k y l l i n k a g e . N o t u n t i l a proton i s t r a n s f e r r e d t othe a c e t a t e oxygen (or to the t h i o a c e t a t e s u l f u r ) does the e l e c t r o n d e n s i t y o f the e s t e r l i n k a g e f a l l to 0.485 (and t h a t o f the t h i o e s t e r l i n k a g e to 0.663), both below the e l e c t r o n d e n s i t y of the adjacent a l k y l l i n k a g e . The e l e c t r o n d e n s i t y o f t h e bond f o r m i n g between t h e a t t a c k i n g m e t h a n o l o x y g e n and t h e s u b s t r a t e carbonyl carbon increases s t e a d i l y f o r a l l proposed intermediates, b u t i s i n i t a i l l y l a r g e r f o r t h e ACh s u b s t r a t e ( 0 . 2 2 0 ) than f o r the ASCh s u b s t r a t e (0.074). A l s o , f o r a l l i n t e r m e d i a t e s , the negative a n t i bonding e l e c t r o n d e n s i t i e s between the a t t a c k i n g methanol o x y g e n and t h e t h i o a c e t a t e s u l f u r and c a r b o n y l o x y g e n are l a r g e r than are the corresponding e l e c t r o n d e n s i t i e s



310


Table IV Bond O v e r l a p

Mechanism Step

Population f o r Acylation Intermediates f o r S u b s t r a t e s ACh e n d ASCh

Alkyl C Acetate 0o r Thioacetate S


ASCh

Reactants IA IIA 11 I A IV

0.579 0.578 0.588 0.528 0.641

0.670 0.673 0.669 0.665 0.692

Mechanism Step

Methanol 0 Carbonyl C

S u b s t r a t e : ACh Reactants IA IIA 111A IV

ASCh

Carbonyl C Acetate 0o r Thioacetate S

Methanol 0 Acetate 0o r Thioacetate S

ACh

ACh

ASCh

0.698 0.765 0.637 0.485

-

0.980 0.893 0.734 0.663

Methanol 0 Carbonyl 0 ACh

0 0 0 0

.074 .499 .665 .708

-0. -0. -0. -0.

146 104 118 186

_

-0.130 -0.101 -0.134

-0.170 -0.157 -0.155

Carbonyl Carbonyl

C 0

1 .,0 2 0 0..841 0,. 6 5 5 0..664 0,. 9 9 5

0..858 0..981 0.. 6 2 3 0.. 6 3 8 0.. 9 9 5

ASCh

_

0. 2 2 0 0. 5 8 8 0. 6 5 3 0. 7 0 8

ASCh

-0. -0. -0. -0.

161 123 128 186


14.

311


coRRiNGTON

f o r the a c e t a t e , h e l p i n g e x p l a i n whythe t o t a l e n e r g i e s f o r the proposed ASCh mechanism s t e p s h i g h e r t h a n f o r t h e c o r r e s p o n d i n g p r o p o s e d ACh steps.

valence were mechanism

Total Overlap Population. While the above study of the i n d i v i d u a l bond o v e r l a p p o p u l a t i o n s i s h e l p f u l i n f o l l o w i n g the changes o c c u r r i n g in the proposed mechanism steps, the t o t a l overlap p o p u l a t i o n , presented in Table V, does not appear to be a u s e f u l v a l u e . T h e t o t a l o v e r l a p p o p u l a t i o n i s the sum over a l l p a i r s o f atoms A and Β o f the o v e r l a p p o p u l a t i o n 0 P defined a b o v e . A l l i n t e r m e d i a t e s and p r o d u c t s have l o w e r t o t a l o v e r l a p than t h e r e a c t a n t s , and t h e a p p a r e n t l y lower energy a c i d c a t a l y z e d pathway does not have the l a r g e s t t o t a l overlap of the three pathways s t u d i e d .


A B

Total Overlap Energy. However, while t o t a l o v e r l a p p o p u l a t i o n does not appear to be a u s e f u l parameter, the calculated total overlap energy, as presented in Table VI, does. T h e t o t a l o v e r l a p e n e r g y i s t h e sum o v e r a l l p a i r s o f atoms A and Β o f t h e o v e r l a p e n e r g y , OE.g, which i s d e f i n e d s i m i l a r l y to the o v e r l a p p o p u l a t i o n above, but elements of the Η-matrix are used i n p l a c e of elements of the o v e r l a p m a t r i x : 0 E

AB

«

4

^ Σ Σ

H..C. C u

j u

w h e r e i a n d j a r e summed o v e r a l l a t o m i c b a s i s o r b i t a l s and u o v e r a l l m o l e c u l a r o r b i t a l s . In b o t h t h e ACh and A S C h a c y l a t i o n m e c h a n i s m s , t h e t o t a l o v e r l a p energy i s a minimum f o r the a c i d c a t a l y z e d pathway, thus corresponds t o the c a l c u l a t e d t o t a l valence energy. T o t a l o v e r l a p energy a l s o becomes more n e g a t i v e as t h e m e c h a n i s m s t e p s p r o c è d e . A n a l y s i s o f the components of the c a l c u l a t e d t o t a l o v e r l a p energy i n d i c a t e s that in the a c i d c a t a l y z e d pathway the i n d i v i d u a l b o n d e n e r g i e s o f t h e l a r g e ACh o r A S C h i n t e r m e d i a t e s were s t a b i l i z e d by the presence of the i m i d a z o l i u m donated p r o t o n more than the i m i d a z o l i u m was d e s t a b i l i z e d , w h i l e i n t h e b a s e c a t a l y z e d p a t h w a y t h e l a r g e ACh o r A S C h i n t e r m e d i a t e s w e r e d e s t a b i l i z e d by t h e a b s e n c e o f t h e p r o t o n d o n a t e d t o t h e i m i d a z o l by t h e a t t a c k i n g m e t h o x y more t h a n t h e i m i d a z o l w a s stabilized. ( T h e o p p o s i t e was o b s e r v e d t o b e t r u e i n d e a c y l a t i o n w h e n t h e a c y l e n z y m e was m o d e l e d a s the s m a l l e r molecule methyl a c e t a t e (14).)



312


Table V Total

Overlap

Mechanism Step

Population

I II III IV

ASCh

-0.423 -0.393 -0.544

t o the

reactants) (C) Base Catalyzed

(B) N o n Catalyzed



(relative

-0.541 -0.591 -0.329

ACh -0.455 -0.377 -0.671 -0.100

ASCh -0.498 -0.402 -0.449 -0.223

ACh

ASCh

-0.909 -0.870

-0.817 -0.802

Table VI Total

Overlap

M e c h a n i sm Step

to reactants)

(B) N o n Catalyzed

(C) Base Catalyzed

ASCh

ACh

ACh

-640 - 4 4 2 -806 - 5 7 9 -816 - 7 2 8

254 121 27 266


S u b s t r a t e : ACh I II III IV

Energy (g-kcal/mole, r e l a t i v e

ASCh 236 123 -29 308

959 881


ASCh 773 700

14.

coRRiNGTON


313


C o n c l u s i ons T h i s s t u d y , e m p l o y i n g ARCANA, a s e m i e m p i r i c a l m o l e c u l a r o r b i t a l c a l c u l a t i o n program, to model the a c y l a t i o n step of the enzymic h y d r o l y s i s mechanism f o r ACh and A S C h i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s i n the M u l l i k e n net a t o m i c c h a r g e s and the o v e r l a p d e n s i t i e s f o r the bonds most i n v o l v e d in the a c y l a t i o n f o r t h e two s u b s t r a t e s a n d t h e p r o p o s e d intermediates. However, t h e s e d i f f e r e n c e s do not seem a s i m p o r t a n t a s the s i m i l a r i t i e s , such a s the l o c a t i o n o f s i m i l a r lowest e n e r g y p i - t y p e empty m o l e c u l a r o r b i t a l s on the p o s i t i v e l y charged carbonyl carbon s u b j e c t i n g i t to n u c l e o p h i l i c a t t a c k , and the s t a b i l i z a t i o n o f s u c h a n a t t a c k b y t h e p r o t o n a t i o n of the n e g a t i v e l y charged carbonyl oxygen. Moreover i n the case o f both s u b s t r a t e s , t h e r e i s s i g n i f i c a n t p a r t i a l pi-bonding in the acetate (or t h i o a c e t a t e ) m o i t y , and p r o t o n a t i o n o f the a c e t a t e o x y g e n (or t h i o a c e t a t e s u l f u r ) i s r e q u i r e d t o d i m i n i s h the e s t e r ( t h i o e s t e r ) l i n k a g e and a l l o w the c h o l i n e ( t h i o c h o l i n e ) t odepart to complete the a c y l a t i o n s t e p . Thus, t h i s study i n d i c a t e s t h a t the a c y l a t i o n step f o r e i t h e r t h e ACh o r t h e A S C h s u b s t r a t e w o u l d p r o c è d e t h r o u g h a low e n e r g y (and thus r a p i d ) a c i d c a t a l y z e d pathway, c o n s i s t e n t with experimental d a t a o n ACh w h i c h suggests that a c y l a t i o n is catalyzed by a f u n c t i o n a l g r o u p o f p K = 5.3 (]J0) a n d w i t h e x p e r i m e n t a l data which show t h a t b o t h ACh and A S C h a r e h y d r o l y z e d a t n e a r l y the same r a t e w i t h t h e s l o w d e a c y l a t i o n s t e p b e i n g the r a t e d e t e r m i n i n g s t e p (5J. Calculation

Note

The t h e o r e t i c a l b a s i s o f t h e m o l e c u l a r o r b i t a l p r o g r a m e m p l o y e d i n t h i s s t u d y , ARCANA, has been developed e l s e w h e r e ( Z j [ , 27) a n d w i l l n o t b e r e p e a t e d h e r e a t l e n g t h . B a s i c a l l y , ARCANA i s a n i n t e r a t i v e e x t e n d e d H u c k l e m e t h o d w h i c h i n c l u d e s n e i g h b o r atom i n t e r a c t i o n s and i t e r a t e s to c h a r g e s e l f - c o n s i s t e n c y . A minimum b a s i s s e t o f i n v a r i e n t b u t o v e r l a p o p t i m i z e d a t o m i c o r b i t a l s (28) a r e e m p l o y e d t o c a l c u l a t e o v e r l a p i n t e g r a l s . The e m p i r i c a l o r b i t a l e n e r g y (29) a n d a n e f f e c t i v e r a d i u s c a l c u l a t e d b y e v a l u a t i n g R. = l / 0 / * \ > f o r a c c u r a t e a b i n i t i o a t o m i c w a v e f u n c t i o n s (30., 3J_) a r e e m p l o y e d t o c a l c u l a t e the Hamiltonian e l e m e n t s . Only the values f o r hydrogen deviate from standard atomic values. R a d i a l moment a n a l y s e s o f a c c u r a t e m o l e c u l a r o r b i t a l s (32) i n d i c a t e d that a contracted, scaled helium-like representation is required f o r hydrogen in a molecule. Table VII summarizes the atomic o r b i t a l parameters employed i n the c a l c u l a t i o n s .


314


Table Orbital Element


Orbital

H

Is

C

2s 2p 2s 2p 2s 2p

Ν 0 a

Parameters n

VII

Employed

ST0

Z

Orbital Energy (eV)

ST0

l

a

2

b

1.57 0.88°

b

1 .88 1.06

1 .45

a

2

l

b

b

ϋ l

2

b

b

2.19K 1.22

R e f . 32_, b R e f . 2 8 , c R e f .

b

- 9.0

a

e

- 1 9 . 5. - 9.9

b

l

i n ARCANA C a l c u l a t i o n s

b

-25.5? -12. 5

C

C

e

-32.0 . -15.3 e

R, = l / < l / r > (a.u.) 1

0.65

a

1.12* 1 .28

d

0.94* 1 .04

d

0.80*J 0.90°

29., d R e f . 3 1

A f t e r i t e r a t i n g t o charge s e l f - c o n s i s t e n c y , a total valence energy i s c a l c u l a t e d . This includes appropriate factors f o r valence electron repulsion (but excludes the core electrons) andincludes nuclear repulsion (but excludes the corresponding core nuclear charges). A recent comparison (33) o f q u a n t i t i e s c a l c u l a t e d from m o l e c u l a r e l e c t r o n i c wave f u n c t i o n s f o r p y r r o l e a n d p y r a z o l e i n d i c a t e d t h a t ARCANA v a l u e s c o m p a r e d more f a v o r a b l y with l a r g e - s c a l e ab i n i t i o values than those c a l c u l a t e d by other non-rigorous methods i nthe case o f orbital energies o foccupied molecular o r b i t a l s , gross atomic populations o f heteroatoms, andtotal overlap populations including negative overlap populations between nonbonded atoms. Acknowledgements The a u t h o r wishes t o thank t h e f o l l o w i n g s t u d e n t r e s e a r c h a s s i s t a n t s f o r t h e i r c o n t r i b u t i o n s t o t h i s work: W a r r e n W e l t e r s , Ramona C a l v e y , D e b r a Simmons, C h e r y l Mackie, Mary Dean, a n dL a r r y Givens. T h i s s t u d y was s u p p o r t e d b y Grant RR-08008 from t h e General Support Branch, D i v i s i o n o f Research Resources, National I n s t i tutes o f Health. Literature Cited

1. Scott, K. A. and Moutner, H. G . , Biochem. Pharmacol., 13, 907 (1964)

2. Mautner, H. G., Bartels, E. and Webb, G. D., Biochem. Pharmacol., 15, 187 (1966)



14.CORRINGTONEnzymic Hydrolysis of Acetylcholine

315

3. Mautner, H. G., J. Gen. Physiol., 54, No. 1, Pt. 2, 2715 (1969) 4. Mautner, H. G., Dexter, D. D . , and Low, B. W., Nature (London), New Biology, 238, 87 (1972) 5. Scott, K. A. and Mautner, H. G . , Biochem. Pharmacol., 13, 907 (1964) 6. Froede, H. C. and Wilson, J . B . , Enzymes, 3rd E d . , 5, 87 (1971) 7. Wilson, I. B. and Bergmann, F., J. Biol. Chem., 186, 683 (1950) 8. Krupka, R. M . , Biochem., 5, 1983 (1966) 9. Krupka, R. M . , Biochem., 5, 1988 (1966) 10. Krupka, R. M . , Biochem., 6, 1183 (1967) 11. Pullman, B. and Courriere, P . , Mol. Pharmacol., 8, 371 (1972) 12. Aldrich, H. S., Int. J. Quantum Chem., Quantum Biol. Symp., 2, 271 (1975) 13. Farkas, M . , Kruglyak, Υ. Α . , Nature (London), 223, 523 (1969) 14. Corrington, J . Η. (submitted) 15. Genson, D. W. and Christoffersen, R. F., J. Amer. Chem. Soc., 95, 362 (1973) 16. Camepa, F. G . , Pauling, P . , and Sorum, H . , Nature (London), 210, 907 (1966) 17. Herdklotz, J . K. and Sass, R. L., Biochem. Biophys. Res. Commun., 40, 583 (1970) 18. Clothia, C. and Pauling, P. J., Nature (London), 223, 919 (1969) 19. Smissman, Ε. E. and Chappell, G. S., J . Med. Chem., 12, 432 (1969) 20. Smissman, E. E., Helson, W. L., LaPidus, J . B. and Day, J. L., J. Med. Chem., 9, 458 (1966) 21. Stephen, W. F., Jr., Smissman, E. E., Schowen, Κ. B . , and Self, G. W., J. Med. Chem., 15, 241 (1972) 22. Schowen, Κ. B . , Smissman, Ε. Ε . , and Stephen, W. F . , Jr., J. Med. Chem., 18, 292 (1975) 23. Shefter, E., Mautner, H. G., and Smissman, Ε. E., Acta Crystallogr. Sect. A, 25, S201 (1969) 24. Rerat, C., Bull. Soc. Franc. Min. Crist., 85, 153 (1962) 25. Mulliken, R. S., J . Chem. Phys., 23, 1833, 1841 (1955) 26. Corrington, J . Η., Aldrich, H. S., McCurdy, C. W., and Cusachs, L. C., Int. J. of Quantum Chem., 5, 307 (1971) 27. Cusachs, L. D. and Miller, D. J., Advan. Chem. Ser. 110, 1 (1972) 28. Cusachs, L. C. and Corrington, J . H . , "Atomic Orbitals for Semiempirical Molecular Orbital Calculations," in Sigma Molecular Orbital Theory, Sinanoglu, O.,


316

29. 30. 31. 32. Downloaded by COLUMBIA UNIV on November 29, 2017 | http://pubs.acs.org Publication Date: November 28, 1979 | doi: 10.1021/bk-1979-0112.ch014

33.

COMPUTER-ASSISTED

DRUG

DESIGN

and Wiberg, Κ. B., e d s . , (Yale University Press, New Haven, Conn., 1970), p. 256 Cusachs, L . C. and Reynolds, J. W., J. Chem. Phys., 43, S160 (1965) Clementi, E., J. Chem., Phys., 41, 295, 303 (1964); J. Chem. Phys., 38, 996, 1001 (1965), IBM J. of Res. and Dev., 9, 2 (1965) Froese, C., J. Chem. Phys., 45, 1417 (1966) and supplement and private communication Cusachs, L . C. and A l d r i c h , H. S . , Int. J . Quantum Chem., 6S, 221 (1972) Kaufman, J. J., Preston, H. J. T., Kerman, E., and Cusachs, L. C., Int. J. of Quantum Chem., 7S, 249 (1973)

Received June 8, 1979.


Theoretical Modeling of Enzymic Hydrolysis of Acetylcholine

Recommend Documents