11 Suicide Enzyme Inactivators
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BRIAN W. M E T C A L F Merrell Research Center, Merrell-National Laboratories, Cincinnati, O H 45215
A new and elegant approach to specific irreversible enzyme inactivation is the use of inhibitors possessing latent reactive functionalities which are unmasked at the enzyme's active site as a result of the normal catalytic turnover. Such an inhibitory process is described by the following equation:
As these inhibitors owe their activity to the k term (i.e., the enzyme's usual mode of action) they have been designated "kcat inhibitors" by Rando (1), while Abeles and Maycock (2) have used the term "Suicide Enzyme Inactivators" because the enzyme, in accepting such a "booby-trapped" substrate commits suicide by its own mechanism of action. An early example of the concept was described by Wood and Ingraham who reported that the product of oxidation of phenol or pyrocatechol by tyrosinase inactivates that enzyme irreversibly(3). It was speculated that the quinonoid products of oxidation react in Michael fashion with nucleophilic residues on the enzyme, leading to covalent binding. cat
What i s now considered as the c l a s s i c a l example o f t h i s con cept was discovered by Endo et a l . (4) who described i n 1970 the i r r e v e r s i b l e i n h i b i t i o n of β-hydroxydecanoylthioester dehydrase by the p r o p a r g y l i c t h i o e s t e r I ( F i g . 1). I , being an analogue of the corresponding c i s o l e f i n , which i s a n a t u r a l s u b s t r a t e , under goes proton a b s t r a c t i o n by the enzyme to generate a p r o p a r g y l i c a n i o n , which on r e p r o t o n a t i o n a f f o r d s the conjugated a l i è n e . This a l i è n e , being a Michael acceptor and hence an a c t i v e a l k y l a t i n g agent, i s able to r e a c t w i t h a n u c l e o p h i l i c h i s t i d i n e residue i n the a c t i v e s i t e of the enzyme leading to covalent bond formation and i r r e v e r s i b l e i n a c t i v a t i o n . During the l a s t nine y e a r s , a number of workers have attempted to g e n e r a l i z e t h i s concept of enzyme i n a c t i v a t i o n to the i n h i b i t i o n o f enzymes other than β-hydroxydecanoylthioester dehy drase. The reader i s r e f e r r e d to e x c e l l e n t reviews by Rando 0-8412-0543-4/80/47-123-241$05.00/0 © 1980 American C h e m i c a l Society Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
C H E M I C A L DETERIORATION O F PROTEINS
A.
Normal enzymatic reactionCH (CH2) CH0HCH C0NAc ^ e
3
CH (CHz)eCH«CHCONAc
2
^
5
CHsfCH^CH'CHCHaCONAc
trans
B.
cis
Inhibition by C H ( C H ) C s C C H 8 C O N A c ' 5
t
5
CHsiCHaJeCiC^HCONAc-»
CH3ÎCH )eCH«C»CHCONAc
NAc*SCH CH NHCOCH 2
Figure 1.
CHsiCHtJeCH^CCHjCONAc
8
2
3
Inhibition of β-hydroxydecanoylthioester
dehydrase
Normal enzymatic reaction · u^Base
A
XCHaCHaCHCOOH ^
NH
XCH CH {CCOOH 2
PyCHo
2
CHjCHtCOCOOH
^
p
S C H
y
C00H
^ J\
XCHJCH-C
/
jJ. ,
®
CH, V"3 l^/COOH
< —
•NH, *'
ipBase —>
2
CH
^
N«CH-Py®
_
Py
^s^COOH N-CH«Py
*
EnzNu
H
HcWcH-C-COOH
N ^ P y ®
N-CH«Py
Figure 2.
—>
H^C^CCC-OH
—>
EnzNu^vs^CCOOH
^ /N - C H « P y
Inhibition of y-cystathionase
Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
NHCH*Py
11.
METCALF
Suicide Enzyme Inactivators
243
Abe les and Maycock (2_) and Walsh (5.) as w e l l as to a symposium proceedings which o f f e r s the most recent and comprehensive reviews i n the area (6). T h i s review w i l l r e s t r i c t i t s e l f to the i r r e v e r s i b l e i n h i b i t i o n of p y r i d o x a l phosphate (PyCHO)-dependent enzymes, a c l a s s of enzymes which has proven to be g e n e r a l l y s u s c e p t i b l e to i n h i b i t i o n by s u i c i d e enzyme i n a c t i v a t o r s . γ-Cystathionase, which c a t a l y z e s the r e a c t i o n shown i n F i g . 2, has the a b i l i t y to c a t a l y z e the a b s t r a c t i o n of both the a- and βprotons of the s u b s t r a t e . In an e a r l y example o f i n h i b i t i o n of a PyCHO-dependent enzyme v i a the s u i c i d e concept, Abeles and Walsh (7) demonstrated that p r o p a r g y l g l y c i n e ( I I ) i s an i r r e v e r s i b l e i n h i b i t o r of γ-cystathionase, which accepts i t as a s u b s t r a t e . The normal proton a b s t r a c t i o n which precedes β - e l i m i n a t i o n induces aliène formation from I I . The aliène, being a Michael acceptor then i n a c t i v a t e s the enzyme v i a an a l k y l a t i v e process i n v o l v i n g a n u c l e o p h i l i c (Nu) residue on the enzyme. P r o p a r g y l g l y c i n e has s i n c e been shown to a l s o i r r e v e r s i b l y i n a c t i v a t e glutamate-pyruvate transaminase (8). Because of i t s p h y s i o l o g i c a l importance, γ-aminobutyric a c i d transaminase (GABA-T), the PyCHO-dependent enzyme r e s p o n s i b l e f o r the catabolism o f the i n h i b i t o r y neurotransmitter, γ-aminobutyric a c i d (GABA) ( F i g . 3 ) , has been subjected to a v a r i e t y of approaches f o r i n h i b i t i o n by s u i c i d e enzyme i n a c t i v a t o r s (9). Ethanolamine-O-sulfate ( I I I ; F i g . 4) was the f i r s t r a t i o n a l l y designed i r r e v e r s i b l e i n h i b i t o r o f GABA-T (10). I l l , being accepted as a s u b s t r a t e i n the same manner as i s GABA, forms a S c h i f f base w i t h PyCHO. In t h i s way, the adjacent C-H bond i s a c t i v a t e d so that proton a b s t r a c t i o n by the enzyme i s f a c i l i t a t e d . The r e s u l t i n g carbanion then induces e l i m i n a t i o n of s u l f a t e and thereby generates an α,β-unsaturated imine which a l k y l a t e s a n u c l e o p h i l i c (Nu) residue i n the a c t i v e s i t e ( F i g . 4 ) . Unfortu n a t e l y , I I I does not r e a d i l y penetrate the b l o o d - b r a i n b a r r i e r and i t s use as a t o o l to study GABA f u n c t i o n has been l i m i t e d . As GABA-T operates by S c h i f f s base-mediated proton a b s t r a c t i o n , γ-acetylenic GABA (IV; F i g . 5 ) , a substrate analogue bear ing an a c e t y l e n i c f u n c t i o n attached to the γ-carbon atom could by analogy t o the i n h i b i t i o n of β-hydroxydecanoylthioester dehydrase by a c e t y l e n i c substrate analogues (4), i r r e v e r s i b l y i n h i b i t t h i s enzyme ( F i g . 5 ) . Thus, r e p r o t o n a t i o n o f the enzymatically-generated p r o p a r g y l i c carbanion could lead to aliène formation. As such an aliène, being conjugated to the imine f u n c t i o n , would be an a l k y l a t i n g agent i r r e v e r s i b l e i n h i b i t i o n should ensue. Based on t h i s premise, γ-acetylenic GABA (IV) was synthe s i z e d (11) and found to be an i r r e v e r s i b l e i n h i b i t o r of GABA-T, i n v i t r o and i n v i v o (12). Thus, when GABA-T, p a r t i a l l y p u r i f i e d from p i g b r a i n , i s incubated f o r v a r y i n g time periods w i t h γa c e t y l e n i c GABA, a time-dependent i n a c t i v a t i o n process i s observed which follows pseudo f i r s t - o r d e r k i n e t i c s . Enzyme h a l f l i v e s range from 28 minutes to 9 minutes with concentrations o f i n h i b i t o r between 0.029 mM and 0.29 mM. Time dependent i n a c t i v a t i o n i s 1
Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
244
C H E M I C A L DETERIORATION O F PROTEINS
Enz-B>
0 III
Nu-Enz
N-CH Py t
N=CHPy
I
0
Nu-Enz
N=CHPy
Elsevier/North Holland Biomedical Press
Figure 4.
Inhibition of γ-aminobutyric
acid transaminase by ethanolamine-O-sulfate
Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
METCALF
Figure 5.
Suicide
Enzyme
Inactivators
Inhibition of γ-aminobutyric acid transaminase by 4-aminohex-5-ynoic acid (γ-acetylenic GABA)
Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
246
C H E M I C A L DETERIORATION
OF PROTEINS
i n d i c a t i v e that covalent m o d i f i c a t i o n has occurred GO and t h i s i s confirmed by the f i n d i n g that prolonged d i a l y s i s of i n h i b i t e d preparations a g a i n s t s e v e r a l b u f f e r s c o n t a i n i n g PyCHO does not r e s t o r e enzyme a c t i v i t y . That the l o s s of enzyme a c t i v i t y i s f i r s t order a t constant i n a c t i v a t o r c o n c e n t r a t i o n i s evidence that i n a c t i v a t i o n occurs before the i n a c t i v a t o r i s r e l e a s e d from the enzyme. When GABA i s added to the i n c u b a t i o n medium, the r a t e o f i n a c t i v a t i o n induced by γ-acetylenic GABA i s d r a m a t i c a l l y reduced. However, when α-ketoglutarate i s a l s o present, t h i s p r o t e c t i o n against i n a c t i v a t i o n i s l o s t . E v i d e n t l y , GABA i s a b l e to protect the enzyme a g a i n s t i n h i b i t i o n because the holoenzyme i s t r a n s formed, i n one turnover, to the pyridoxamine form. This cannot b i n d the i n h i b i t o r as S c h i f f ' s base formation i s no longer p o s s i b l e . In the presence of α-ketoglutarate, the p y r i d o x a l form i s regenerated and i n h i b i t i o n can ensue. γ-Acetylenic GABA thus appears to be a s u i c i d e i n a c t i v a t o r of GABA-T as most of the k i n e t i c c r i t e r i a (2) which are i n d i c a t i v e of substrate-induced i r r e v e r s i b l e enzyme i n a c t i v a t i o n are s a t i s f i e d . I n h i b i t i o n o f PyCHO-dependent enzymes by β,γ-unsaturated amines i s not l i m i t e d to e x p l o i t a t i o n of carbanion-induced a c e t y l e n e - a l l e n e isomerism. As demonstrated i n F i g . 6, a l l y l amines can a l s o i r r e v e r s i b l y i n a c t i v a t e PyCHO-dependent enzymes v i a mechanisms i n v o l v i n g double bond isomerism. Thus, i f Y - v i n y l GABA (V) were a s u b s t r a t e f o r GABA-T, the normal transamination mechanism (path a) would lead to a conjugated imine. A l t e r n a t i v e l y (path b ) , isomerism of the double bond would generate a new double bond, which would be conjugated through t o the p y r i d i n e r i n g . In e i t h e r case, an a l k y l a t i n g agent would be formed as a r e s u l t of the enzyme's own mode of a c t i o n . The transamination pathway i s the one which has been found by Rando et a l . (13) to be o p e r a t i v e i n the i r r e v e r s i b l e i n h i b i t i o n of a s p a r t a t e amino t r a n s f e r a s e by 2-amino-4-me thoxy-trans-3-butenoic a c i d . On the other hand, α-vinyl g l y c i n e i n h i b i t s the same enzyme v i a the isomerism pathway (14). As a n t i c i p a t e d , γ-vinyl GABA (V) i s an i r r e v e r s i b l e i n h i b i t o r of GABA-T i n v i t r o (15) and i n v i v o (16) although whether i n h i b i t i o n occurs v i a transamination (path a) or isomerism (path b) i s as yet unknown. In p r i n c i p l e , enzyme i n h i b i t o r s which r e q u i r e transformation by the t a r g e t enzyme p r i o r t o that enzyme's i r r e v e r s i b l e i n h i b i t i o n should be extremely s p e c i f i c as they should i n h i b i t o n l y those enzymes which can accept them as s u b s t r a t e s . In keeping w i t h t h i s , γ-acetylenic GABA (IV) has l i t t l e e f f e c t on a l a n i n e and asparate aminotransferases. However, i t has now been found t o be an i r r e v e r s i b l e i n h i b i t o r o f the PyCHO-dependent glutamic a c i d decarboxylase (GAD) (17) and o r n i t h i n e aminotransferase (OAT) (18). Since GABA i s a s u b s t r a t e f o r OAT, i n h i b i t i o n o f t h i s enzyme by γ-acetylenic GABA (IV) i s not s u r p r i s i n g . I n h i b i t i o n of GAD by IV was unexpected and w i l l be discussed l a t e r . γ-Vinyl GABA (V) on the other hand, appears to be the most s p e c i f i c i n h i b i t o r o f GABA-T known. To date, no other enzyme has been found to be
Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
11.
M E T C A L F
247
Suicide Enzyme Inactivators
NHCH Py®
(VcH=Py
NH,
