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Inactivation of α-chymotrypsin by new bifunctional reagents : halomethylated derivatives of dihydrocoumarins
M6moires originaux
BIOCHIMIE, 1977, 59, 231-239.
Inactivation of -chymotrypsin by new bifunctional reagents halomethylated derivatives of dihydrocoumarins. Jean-Jacques
B~CHET ~ , A l a i n DUPAIX a n d I v a BLAGOEVA ~.
Laboratoire d ' E n z y m o l o g i e P h y s i c o - c h i m i q u e et Mol~culaire, Groupe de R e c h e r c h e s du CNRS associ~ it rUniversit~ de Paris-Sad, Orsay, 91~05, France. (29-10-1976). Summary. - - ~ - C h y m o t r y p s i n is r a p i d l y a n d completely i n a c t i v a t e d by a series of h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s at pH 7 a n d 25 °. The i n a c t i v a t i o n is p H - d e p e n d e n t a n d o p t i m a l at n e u t r a l pH ; it is also more or less complete depending on the excess of i n h i b i t o r w i t h respect to t h e enzyme. These compounds are s u b s t r a t e s for ~ - c h y m o t r y p s i n and, d u r i n g the c a t a l y t i c process, a l a t e n t a l k y l a t i n g f u n c t i o n of the reagent is activated at the active site of the e n z y m e a n d reacts w i t h some vicinal nueleophilic a m i n o acid residues. The s t o i c h i o m e t r y of the r e a c t i o n of the corresponding radioactive reagents w i t h the e n z y m e is slightly superior to one, b u t one h i s t i d i n e residue of a - e h y m o t r y p s i n is m a i n l y modified. This h i s t i d i n e is identified as histidine-57 b y the d i a g o n a l peptide m a p p i n g method. In c o m p a r i s o n 'with o t h e r reagents, the efficiency of these ¢ suicide s u b s t r a t e s ~ a n d p a r t i c u l a r l y t h a t one of a derivative (compound 2) c a r r y i n g a s u b s t r a t e - l i k e side e h a i n is f o u n d to be very high.
Tile s e l e c t i v e m o d i f i c a t i o n of a m i n o a c i d s p r e s e n t i n t h e a c t i v e s i t e s of e n z y m e s is o f t e n o b t a i n e d b y t h e u s e of a c t i v e - s i t e - d i r e c t e d r e a g e n t s [1]. These reagents have substrate-like structural f e a t u r e s ' w h i c h p r o m o t e t h e f o r m a t i o n of a n adsorption complex with the enzyme ; a chemical g r o u p of t h e r e a g e n t m a y t h e n a c h i e v e a c o v a l e n t m o d i f i c a t i o n w i t h i n t h e a c t i v e site. T h u s p r o t e a s e s may be specifically alkylated by peptide chlorom e t h y l k e t o n e s [2, a n d r e f e r e n c e s t h e r e i n ] . The use of substrates carrying a latent chemical g r o u p a c t i v a b l e i n t h e a c t i v e s i t e of a n e n z y m e d u r i n g t h e c a t a l y t i c p r o c e s s , m a y also l e a d to s o m e i n f o r m a t i o n o n t h e n a t u r e of d i f f e r e n t a m i n o acid residues present in this active site and their respective accessibility and reactivity. This kind of inhibitor has been recently reviewed by Rando [3] a n d S i g m a n a n d M o o s e r [4] a n d h a s b e e n def i n e d as < > o r <> U ) .
T h e h a l o m e t h y l a t e d d e r i v a t i v e s of d i h y d r o c o u m a r i n s ( l i k e c o m p o u n d 1 i n f i g u r e 1) a r e p o t e n tially such bifunctional reagents for esterases and proteases having an esterolytic activity. These compounds possess a reactive ester bond ~-ith a cis c o n f i g u r a t i o n , a n d a n a I k y l a t i n g f u n c t i o n
XCH2
1
1 ~ X=CI 5,
Br
X CH2
Br
3,
Abbreviations used are : CT, (x-chymotrypsin ; AcTyrOEt, N-acetyl-L-tyrosine ethyl e s t e r ; Mes, 2-(Nm o r p h o l i ~ o ) e t h a n e s u l f o n i c acid ; Hepes, N-2-hydroxye t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid. Enzymes : c h y m o t r y p s i n (EC 3.4.4.5) ; pepsin (EC 3.4.4.1). (*) This a p p e l l a t i o n of <> m a y be f o u n d a m b i g u o u s ; the referee suggests the expression <> for such reagents. <> To 'whom all correspondence should be addressed. P r e s e n t a d d r e s s : L a b o r a t o i r e de Biologie Physicochimique, Universitd de Paris-Sud, Orsay, 91405, France. -~ P e r m a n e n t address : I n s t i t u t de Chimie Organique, Sofia 13, Bulgarie.
X=Br
6 ~X=H
X=B,
BrCH2
4
7~X=H FIG.
1. --
Structure of compounds.
w h i c h a p p e a r s a f t e r h y d r o l y t i c c l e a v a g e of t h e ester bond. Indeed the appearing p-hydroxybenzyl h a l i d e is a c t i v a t e d i n t o q u i n o n e m e t h i d e w h i c h reacts very rapidly with any neighboring nuc l e o p h i l i c r e s i d u e [5, a n d r e f e r e n c e s t h e r e i n ] . 18
J.-J. Bdchet, A. Dupaix and I. Blagoeua.
232
It has been p r e v i o u s l y s h o w n that 3,4-dih y d r o 3 , 4 - d i b r o m o - 6 - b r o m o m e t h y l c o u m a r i n (comp o u n d 3), thanks to these features, q u i c k l y inactivated (~-chymotrypsin ES]. H o w e v e r this d i h y d r o c o u m a r i n was r a t h e r u n s t a b l e i n aqueous solution because of a s p o n t a n e o u s d e h y d r o h a l o g e n a t i o n r e a c t i o n on its c a r b o n s 3 a n d 4. Therefore the more simple d i h y d r o c o u m a r i n 1 a n d its analogue 2 c a r r y i n g a substrate-like b e n z y l side c h a i n have n o w been p r e p a r e d b y W a k s e l m a n el al. E6, 7] i n the L a b o r a t o i r e de Chimie Organique Biologique d'Orsay. We study in this p a p e r t h e i r effects u p o n (t-chymotrypsin a n d c o m p a r e them to those of the acyl-activated d i h y d r o c o u m a r i n 3. I n the accomp a n y i n g p a p e r E8~, the action of these reagents t o w a r d s various proteases a n d esterases is reported. EXPERIMENTAL PROCEDURE. MATERIALS.
a - C h y m o t r y p s i n was a salt-free, three-times crystallized p r o d u c t p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l Corp. It was dissolved i n 0.001 N HC1 a n d kept at 4 ° before use. Its c o n c e n t r a t i o n in solution was s p e c t r o p h o t o m e t r i c a l l y d e t e r m i n e d at 280 n m u s i n g a m o l a r e x t i n c t i o n coefficient of 5 × 104 [9] a n d a m o l e c u l a r weight of 2.5 × 104 [10]. P e p s i n was a twice-crystallized p r o d u c t , purchased from W o r t h i n g t o n Biochemical Corp. N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) was o b t a i n e d from Mann Research Laboratories. The buffers used w e r e N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid (Hepes)-NaOH a n d 2 - ( N - m o r p h o l i n o ) - e t h a n e s u l f o n i c acid (Mes)-NaOH whose sulfonic acids w e r e p u r c h a s e d from Calbiochem Chemical Co. Acetate, borate a n d phosphate buffers w e r e p r e p a r e d from P r o l a b o products. Acetonitrile w a s purified a c c o r d i n g to Coetzee !11]. PREPARATION AND CHARACTERISATION OF DIHYDROCOUMARINS AND COUMARINS.
Most of the c o m p o u n d s w e r e synthesized a n d c h a r a c t e r i z e d as r e p o r t e d p r e v i o u s l y [5, 6~. The d i h y d r o c o u m a r i n s 2 a n d 5 have been r e c e n t l y p r e p a r e d ET]. Some special care was taken d u r i n g the use of the d i h y d r o c o u m a r i n 2 w h i c h m a y be toxic to h u m a n s because of possible a n t i c o a g u l a n t p r o p e r t i e s E7]. The syntheses of the r a d i o a c t i v e d i h y d r o c o u m a r i n s [14CJ 1 a n d [~H] 3 have been d e s c r i b e d b y W a k s e l m a n el al. [6]. The specific r a d i o a c i i vities of [I4C] 1 and IaH] 3 c a r r y i n g the radio-
BIOCHIMIE, 1977, 59, n ° 3.
active atoms on the c h l o r o m e t h y l group was 0.5 I~Ci/mM a n d 730 ~Ci/mM, respectively. The r a d i o a c t i v e derivative EI~C] 2 could not be prepared. The ultra-violet a b s o r p t i o n spectra of the differ e n t d i h y d r o c o u m a r i n s in e t h a n o l w e r e r e c o r d e d before each use of the c o m p o u n d s ; they w e r e characterized by small peaks b e t w e e n 290 n m a n d 260 n m and a strong increase of the a b s o r b a n c e between 200 n m and 240 n m : for 1, ~ma~ (EtOH) 270 n m (e 750 M-1 cm -1) a n d 279 n m (e 610 M-1 cm -1) ; for 2, )~n~ax(EtOH) 265 n m (e 950 M-1 cm-1), 268 n m (e 910 M-1 cm-1) a n d 280 n m (e 570 M-1 cm -1) ; for 3, k (EtOH) 285 n m (shoulder, 1900 M-1 cm-1) ; for 5, kmax (EtOH) 272 n m (~ 990 M-1 cm-1) a n d 280 n m (e 890 M-1 cm-1) ; for 6, k.... (EtOH) 280 n m (e 925 M-1 cm -1) a n d 272 n m (e 1010 M-1 cm-I) ; for 7, )~ (EtOH) 290 n m (shoulder, ~ 2~080 M-~ c m l i n s t e a d of 2700 M~1 cin-L at 287 nm, as p r e v i o u s l y r e p o r t e d [5] for a p r o d u c t slightly decomposed). METHODS.
Reactions of the dihgdrocoumarins with ¢x-chgmotrgpsin. 10-100 i~l of a freshly p r e p a r e d solution of 10 mM d i h y d r o c o u m a r i n in acetonitrile were added w i t h s w i r l i n g to a solution of (t-chymot r y p s i n (at final c o n c e n t r a t i o n s 4-30 ,~M) in 5 ml of 25 mM Hepes-NaOH or Mes-NaOH buffers (0.5 M NaC1 ; final p e r c e n t of acetonitrile, 0.2-2), at a given pH a n d 25 °. After different i n c u b a t i o n periods w i t h i n one hour, the c h y m o t r y p t i c activity of a small aliquot w a s tested against AcTyrOEt (10 mM or 5 raM) i n 10 ml of 0.1 M NaC1 at pH 7.5 a n d 25 °. The e n z y m a t i c h y d r o l y s i s of AcTyrOEt was followed b y pH star m e a s u r e m e n t s u s i n g a Vibron p H m e t e r model 33 B (Electronic I n s t r u m e n t s Ltd). In the same e x p e r i m e n t a l c o n d i t i o n s a n d in the absence of reagent, it was verified that the enzyme did not s p o n t a n e o u s l y lose significant activity.
Spontaneous and enzymatic hgdrolgses of dihgdrocoumarins. Kinetic m e a s u r e m e n t s w e r e c a r r i e d out at 25 ° in aqueous solutions (0.5 M NaC1) c o n t a i n i n g 1 p e r cent (v/v) acetonitrile. E m p l o y e d buffers were 0.025 M Mes-NaOH b e t w e e n pH 6 and 7 ; 0.025 M Hepes-NaOH b e t w e e n p H 7 a n d 8 ; 0.05 M b o r i c a c i d - b o r a x b e t w e e n pH 8 a n d 9 ; 0.0125 M boraxNaOH b e t w e e n pH 9 a n d 10. The s p o n t a n e o u s a n d enzymatic h y d r o l y s e s of the d i h y d r o c o u m a r i n s 1 a n d 6 w e r e followed b y s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s w i t h a Cary 16
Inactivation of a-chymotrypsin by <>. s p e c t r o p h o t o m e t e r e q u i p p e d w i t h a thermostated cell c o m p a r t m e n t and coupled to a Sefram Grap h i s p o t recorder. The h y d r o l y s i s of the lactone 1 was followed at 286 n m ( a p p e a r a n c e of the product) or at 2'50 n m ( d i s a p p e a r a n c e of the subs t r a t e ; p a r t i c u l a r l y , at pH values lower t h a n 8). The respective m o l a r a b s o r b a n c e changes were equal to 825 M-1 cm q a n d 3500 M-1 cm 1 (at least, at p H values lower t h a n 9 w h e r e the i o n i s a t i o n of the p h e n o l f u n c t i o n of the p r o d u c t is negligible). The h y d r o l y s i s of the analogue 6 ~vas followed at 286 n m (Ae 1685 M-1 cm-1). At a n y pH, the pseudo first-order rate constants for the spontaneous h y d r o l y s e s of lactones 1 or 6 were calculated from the slopes of log [ODoc- O D o ) / ( O D ~ ODt)] versus time t. F o r the evaluation of the kinetic constants K m a n d keat i n the ct-chymotrypsin-catalyzed h y d r o lysis of 6 at 2'5~' a n d pH 7.5 (0.025 M Hepes-NaOH ; 0.5 M NaC1; 3.8 per cent (v/v) acetonitrile), the substrate c o n c e n t r a t i o n was varied b e t w e e n 0.19 mM a n d 1.9 m M ; the enzyme c o n c e n t r a t i o n w a s equal to 0.018 mM in both sample a n d refer e n c e cells. The n o n e n z y m a t i c h y d r o l y s i s rate was also m e a s u r e d a n d the necessary c o r r e c t i o n s were made. The d i h y d r o c o u m a r i n 6 (at the c o n c e n t r a t i o n 0.45 m M ) w a s also tested as a competitive i n h i b i tor in the a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of AcTyrOEt, at pH 7.5 a n d 25 °. The m e a s u r e m e n t s were p o t e n t i o m e t r i c ; the u n b u f f e r e d m e d i u m c o n t a i n e d 0.5 M NaC1 a n d 1 per cent (v/v) acetonitrile. The substrate c o n c e n t r a t i o n was v a r i e d b e t w e e n 0.2 mM a n d 9.5 raM. The Micha~lis c o n s t a n t Km for the e n z y m a t i c h y d r o l y s i s of AcTyrOEt was d e t e r m i n e d in the p r e s e n c e and absence of d i h y d r o c o u m a r i n , a n d the i n h i b i t i o n c o n s t a n t K i was deduced from the different values of K,~.
Preparation of the inactivated ¢t-chymotrypsin. c¢-Chymotrypsin (0.4-0.6 m g / m l ) was i n c u b a t e d in the p r e s e n c e of a large excess of reagent ([1] = 0.5 mM or [21 ~ 0.07 mM or [3] ~ 0.2 raM), in a given volume (5 ml-250 ml) of 0.025 M HepesNaOH buffer at pH 7 a n d 25 ° for 30 m i n u t e s (with the reagent 1) or 15 m i n u t e s (with the reagents 2 or 3). The e n z y m a t i c activity was m e a s u r e d before a n d after i n c u b a t i o n . Then, the pH value of the solution w a s adjusted to pH 3 ~with HC1 or formic acid (not in the case of the enzyme i n a c t i v a t e d by 3). The modified ¢t-chymotrypsin ~ a s either filtered t h r o u g h Sephadex G 25 e q u i l i b r a t e d w i t h 0.001 N HC1 at 4 °, or exhaustively dialyzed w i t h twice-boiled dialysis bags against 0.001 N HC1 at 4 ° d u r i n g 48 or 72 h o u r s if large volumes of solu-
BIOCHIMIE, 1977, 59, n ° 3.
233
tion were nsed. I n this last case, the modified enzyme was ultrafiltrated a n d c o n c e n t r a t e d on Diaflo PM-10 or UM-10 m e m b r a n e (Amicon Corp.). F i n a l l y the enzyme solution was passed t h r o u g h a 0.22 .!xin Millipore filter and stored at 4 °. (t-Chymotrypsin modified by the r a d i o a c t i v e derivative E14C] 1 was p r e p a r e d in the same m a n n e r as described above and its r a d i o a c t i v i t y was d e t e r m i n e d as p r e v i o u s l y reported ES].
Amino acid analyses. Native or modified a - c h y m o t r y p s i n was at first filtered t h r o u g h Sephadex G-25 e q u i l i b r a t e d w i t h 0.001 N HCI. Samples of p r o t e i n (1-1.5 rag) were then lyophilized and, occasionally, p e r f o r m i c acid oxidized [12] a n d lyophilized again. T h e y ~-ere m i x e d w i t h c o n s t a n t - b o i l i n g HC1, deaerated a n d h y d r o l y z e d in vacuo for 22 hours at 110 °. Hydrolysates were evaporated to d r y n e s s and the residues taken up i n 0.2 M s o d i u m citrate buffer pH 2.2. Amino acid analyses were p e r f o r m e d by the methgd of S p a c k m a n el al. I13] with a Beckm a n Multichrom automatic a m i n o acid a n a l y s e r coupled with an I n f o t r o n i c s model C.R.S. 210 integrator. Molar ratios of a m i n o acids were calculated on the basis of l e u c i n e ---- 19.
Diagonal paper electrophoresis. Diagonal p a p e r electrophoretograms of native or i n h i b i t e d c h y m o t r y p s i n s were p r e p a r e d accord i n g to the method of B r o w n a n d Hartley [14]. T y p i c a l l y , the native or modified c h y m o t r y p s i n (50 rag) was digested w i t h p e p s i n (5 mg) i n 10 ml of 5 per cent formic acid for 18 hours at 37 °. The peptic digest was spotted (1 m g / c m ) as a 3-cm wide b a n d in the center of a sheet of W h a t m a n 3MM paper. It was then subjected to high-voltage electrophoresis p e r f o r m e d w i t h a Gilson F r a n c e model D a p p a r a t u s u s i n g varsol coolant a n d a buffer of adequate pH, at 3000 V at pH 6.5 for 1 h o u r (or 50 rain in the first e x p e r i m e n t s w i t h the native enzyme). A 3-cm w i d e strip was cut from the edge of the dried sheet and exposed for 2 h o u r s at room t e m p e r a t u r e in the vapors of p e r f o r m i c acid. After drying, the oxidized strip was sewn across the center of a n o t h e r sheet of W h a t m a n 3MM p a p e r and subjected again to highvoltage electrophoresis at pH 6.5 at 3000 V for 1 hour. The peptides were detected with the cadm i u m - n i n h y d r i n reagent [15] a n d those contain i n g h i s t i d i n e w i t h the P a u l y reagent [16]. After e x a m i n a t i o n of the o b t a i n e d d i a g o n a l p e p t i d e map, the p u r i f i c a t i o n of p a r t i c u l a r cysteic acid peptides called A'I a n d A'2 (see results) was made in the following way. The cystine p e p t i d e p a r e n t of A'I a n d A'2 was cut out as a b a n d from
J.-J. B d c h e t , A . D u p a i x a n d I. B l a g o e v a .
234
the first electrophoretogram and oxidized for 2 h o u r s at r o o m t e m p e r a t u r e i n t h e v a p o r s of p e r fornfic acid. After drying, the oxidized strip was s e w n o n to a s e c o n d s h e e t of W h a t m a n 3MM paper and the peptides were then purified by h i g h - v o l t a g e e l e c t r o p h o r e s i s a t 4500 V a t p H 6.5 f o r 45 m i n u t e s . C y s t e i c a c i d p e p t i d e s ' w e r e d e t e c ted on side strips with the cadmium-ninhydrin r e a g e n t a n d e l u t e d w i t h 0.1 M NH~. A f t e r e v a p o r a t i o n to d r y n e s s , p e p t i d e s w e r e h y d r o l y z e d i n 5.7 N HC1 i n s e a l e d e v a c u a t e d t u b e s f o r 22 h o u r s at 110 ° f o r a m i n o a c i d a n a l y s e s .
i n f i g u r e 4, a s l i g h t e x c e s s of a b o u t 2-3 t i m e s of t h e r e a g e n t 2 o n t h e e n z y m e is s u f f i c i e n t to i n a c t i vate a-chymotrypsin completely, while an excess of 20-30 t i m e s of t h e r e a g e n t 1 o n t h e e n z y m e is r e q u i r e d to o b t a i n t h e s a m e r e s u l t .
10(
~
CJ
t~75
~
[_0
[
' '
19,65.M
>-
J = 35 . M
RESULTS.
<
E f f e c t of p H on the inactivation of ~J.-chymotrypsin by h a l o m e t h y l a t e d derivatives of dihydrocoumarins. A t n e u t r a l p H , t h e d i h y d r o c o u m a r i n s 1, 2 a n d 3 ( i n e x c e s s of 10-40 t i m e s o n t h e e n z y m e ) i n a c t i vate completely and irreversibly a-ehymotrypsin within a few minutes. However, at more aeidic p H v a l u e s , t h e e n z y m e is less c o m p l e t e l y a n d q u i c k l y i n a c t i v a t e d (figure 2). T h i s s a m e p h e n o m e n o n is o b s e r v e d f o r all t h r e e r e a g e n t s .
i2~
I :
0
=
~
A"
[
10
20
.
.
=,
30 40 TIME ffmnl
z 75
pH 5.55
•
pH 6.05
'
-
•
0
O
10
20
30 40 TIME I m m )
50
i
i
60
70
FIG. 2. - - Effect of pH on the inhibition of a-chymotrypsin by 3,$-dihydro-6-chloromethylcoumarin 1 at 25°: (©) pH 5.55 ;(O) pH 6.05; (El) pH 6.45; (n) pH 7.05 ; (A) pH 7.45; (A) pH 8.3. The enzyme concent r a t i o n 'was 4.95 uM ; the reagent c o n c e n t r a t i o n was 96 ittM i n 0.025 M Hepes-NaOH or Mes-NaOH buffers (0.5 M NaC1 ; 1 per cent (v/v) acetonitrile).
of d i h g d r o c o u m a r i n s .
E f f e c t of reagent concentration on the extent of inactivation. At a g i v e n p H v a l u e , t h e r e s i d u a l a c t i v i t y o b s e r v e d a t t h e e n d of t h e r e a c t i o n d e p e n d s o n t h e m o l a r r a t i o of i n h i b i t o r to e n z y m e a n d a n a d e q u a t e e x c e s s of i n h i b i t o r is n e c e s s a r y to p r o d u c e a c o m p l e t e i n a c t i v a t i o n ( f i g u r e 3). Moreover this phenomenon depends on the s t r u c t u r e of t h e d i h y d r o c o u m a r i n . T h u s as s h o w n
0
75
'/ 2
3
1
~_50 z
--25
BIOCHIMIE, 1977, 59, n ° 3.
7=0
-
100
pH 7,05
m
6=0
In fact, dihydrocoumarins are substrates for a - c h y m o t r y p s i n . T h u s , t h e k i n e t i c c o n s t a n t s K m, kca t a n d k,.at/K m f o r t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of t h e s i m p l e s t 3 , 4 - d i h y d r o - 6 - m e t h y l coumarin 6 ('which does not irreversibly inactiv a t e t h e e n z y m e ) a r e e q u a l to 0.22 mM, 0.021 s e c -I a n d 95 M q s e c -1 r e s p e c t i v e l y , at p H 7.5 a n d 25 °.
0
50
510
FIG. 3. Effect of reagent concentration on the percentage inhibition of ~-chymolrypsin by 3,$-dihydro-6-chloromethylcoumarin 1 at pH 7.05 and '25°. The e n z y m e c o n c e n t r a t i o n w a s 4.95 IvM a n d the concent r a t i o n of the i n h i b i t o r I 'was v a r i a b l e : (©) no reagent ; (O) 19.65 ~tM ; ([3) 35 ~tM ; (ll) 50 ~M ; (~) 95 I~M ; (&) 195 uM.
E n z g m a t i c hgdrolgsis 00
50 . M
La
~
O0
•
5
10 [INHIBITOR]/IENZYMEi
15
'~0
Fro. 4. - - Percentage inhibition as a function of the molar ratio of dihydrocoumarine 1,2 or 3 Iwith respect to u-chymotrypsin at pH 7 and 25 ° : (0), c o n c e n t r a t i o n of the reagent I = 19.3 aM-196 UM, c o n c e n t r a t i o n of the e n z y m e = 4.93 lu.M, i n c u b a t i o n t i m e = one h o u r ; (@), c o n c e n t r a t i o n of the r e a g e n t 2 : 5.87 ,~M - 68.2 ~M, e n z y m e c o n c e n t r a t i o n = 10.9 ~M, i n c u b a t i o n t i m e 15 m i n u t e s ; (A), c o n c e n t r a t i o n of the reagent 3 ----24 i~M- 185 ,~tM, enzyme c o n c e n t r a t i o n = 25 ~tM, incub a t i o n t i m e ---- 15 minutes.
Inactivation of a - c h g m o t r g p s i n Furthermore, this titive i n h i b i t o r in specific substrate constant Ki equal Km value.
l a c t o n e 6 b e h a v e s as a c o m p e the e n z y m a t i c h y d r o l y s i s of the AcTyrOEt, with an inhibition to 0.2 mM a n d i d e n t i c a l to t h e
I n c a s e of t h e m o r e s p e c i f i c r e a c t i o n w i t h t h e hindered dihydrocoumarin 2, ~ - e h y m o t r y p s i n turns over between one and two molar equivalents o f t h e c o m p o u n d 2, b e f o r e it b e c o m e s c o m p l e t e l y i n a c t i v a t e d (figure 4 ) ; t h e l o w s o l u b i l i t y of
T h e d i h y d r o c o n m a r i n 1 is also h y d r o l y z e d b y a - c h y m o t r y p s i n (*) at a r a t e s o m e w h a t h i g h e r t h a n 6, b u t t h e e n z y m a t i c r e a c t i o n b e c o m e s n e g l i gible in a few m i n u t e s b e f o r e c o m p l e t e h y d r o l y s i s o f t h e s u b s t r a t e , as s h o w n i n figure 5. F o r t h e r e a c t i o n of 1 (in a s u f f i c i e n t e x c e s s ) w i t h ¢t-chym o t r y p s i n at p H 7.5 a n d 25 ° , t h e e n z y m e t u r n s over about 6 m o l a r e q u i v a l e n t s of 1 b e f o r e complete inactivation. For comparison, the slower and more continuous trypsin-catalyzed hydrolysis of this d i h y d r o c o u m a r i n is also s h o w n in f i g u r e 5 ; as r e p o r t e d i n t h e a c c o m p a n y i n g p a p e r [8], t h e i n a c t i v a t i o n o f t r y p s i n b y 1 is r e l a tively slow and uncomplete. (% It has been verified t h a t the rates of the spontaneous h y d r o l y s e s of the d i h y d r o c o u m a r i n s 1 and 6 were relatively negligible 'with regard to those of their enzymatic hydrolyses, at pH 7.5 and 25 ° . In the alkaline pH range 7.5-9.5, the second-order rate constants ko~- for the alkaline hydrolyses of compounds 1 and 6 are 310 M-1 see-1 and 170 M-1 sec-L respectively. Between pH 6 and 8, some w a t e r - c a t a lyzed hydrolysis of compound 1 is observed ~vith a first-order rate c o n s t a n t ko equal to 1.5 X 10-4 sec-1 (spectrophotometric m e a s u r e m e n t s at 250 rim).
235
by ¢ suicide s u b s t r a t e s >~.
i,'.. °"°"°*°°
E =0
,3 E : 29.5~M
TIME (mm)
FIG. 5. - - K i n e t i c runs of the a-chymotrypsin-catalyzed hydrolysis of dihydrocoumarin 1 at pH 7.5 and 25°; spectrophotometric m e a s u r e m e n t s ; reagent conc e n t r a t i o n = 196 j~M ; variable enzyme concentration, E. A kinetic run of the t r y p s i n - c a t a l y z e d h y d r o l y s i s of d i h y d r o c o u m a r i n 1 at pH 7.5 and 25" is also sho~vn ( @ Q O O ) : reagent concentration = 196 uM ; enzyme c o n c e n t r a t i o n = 14.1 ~M. The enzyme w a s p r e s e n t in both sample and reference cells. The final absorbance of the h y d r o l y s i s product of 1 is also given (. . . . . ).
TABLE I.
Amino acid composition of native a-chymotrypsin and a-chymotrypsin modified by halomethylated derivatives of dihydrocoumarins (a). Native C T Amino acid
I
Theoretical values (b)
Experimental values
C T modified (3 T modified by 2 by I
C T modilied by 3 (c }
I Aspartic acid . . . . . . . . . Threonine (d) . . . . . . . . Serine (d) . . . . . . . . . . . Glutamic acid . . . . . . . Proline . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . Half cystine (e) . . . . . ¥aline . . . . . . . . . . . . . . . Methionine . . . . . . . . . . .
22 22 27 15 9 23 22 10 23 2
Isoleucine . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . Phenylalanine . . . . . . . . Lysine . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . Arginine . . . . . . . . . . . . .
10 19 4 6 14 2 3
22 18.3 185 15.3 89 23.3 22.2 10.2 22.75 1.95 (f) 1.6(g) 9.4 19 3.9 5.7 14.3 1.95 3.05
22.4 18.2 18.2 15.45 9.2 24.1 22.6 10 23.45 1.95 (I) 1.45 (g) 9.45 19 4 5.9 13.9 1,25 2.9
22.7 18.55 18.65 15.3 9.0 23.1 22.1 10 22.85 2.2 (f) 1.65 (g) 9.4 19 3.9 5.8 13.85 1.1 2.95
21.3 21.3 24.8 16.5 9.8 23.2 22.5 21 2 9.7 19 3.3 64 14.2 1.0 3.1
(a) The m e a s u r e d values are the average of five analyses. (b) F r o m Dayhoff [40]. (c) F r o m B6chet et al. [15]. (d) Not corrected for h y d r o l y t i c destruction. (e) Determined as eysteic acid a f t e r p e r f o r m i c o x y d a t i o n and corrected for 94 per cent recovery [4,1]. (f) Sum of m e t h i o n i n e and m e t h i o n i n e sulfoxides. (g) Determined as m e t h i o n i n e sulfone after performic oxydation and not corrected for o v e r o x y d a t i o n [41].
BIOCHIMIE, 1977, 59, n ° 3.
236
J.-J. Bdchet, A. D u p a i x and I. Bla~!oeva.
the reagent did not h o w e v e r measurement.
allo'w its direct
Sloiehiometry of the inactivation reaction. a - C h y i u o t r y p s i n i n c u b a t e d i n the p r e s e n c e of the d i h y d r o c o u m a r i n s 1, 2 a n d 3 i n a large excess at pH 7 is i r r e v e r s i b l y i n a c t i v a t e d a n d does not recover significant activity after dialysis a n d t r e a t m e n t w i t h 1 M h y d r o x y l a m i n e at pH 7.5 a n d 20 ° for 24 hours. I n c u b a t i o n e x p e r i m e n t s ~¢ith radioactive derivatives [14CJ 1 a n d [3H] 3 [ref. 5] in excess at pH 7 and 25 ° show that 1.3 _+ 0.1 a n d 1.25 _+ 0.1 molecules of the labeled reagent are i n c o r p o rated per molecule of protein. W h e n the enzymes has been p r e v i o u s l y completely i n h i b i t e d b y p h e n y h n e t h a n e s u l f o n y l fluoride w h i c h reacts w i t h the active serine 195 p17, 18], a negligible i n c o r p o r a t i o n of the reagent [14C] 1 i n the p r o t e i n (about 0.08 molecules of reagent per molecule of p r o t e i n ) is observed).
Amino acid analyses of the inactivated enzyme. A m i n o acid analyses of a - c h y m o t r y p s i n , before and after i n a c t i v a t i o n by the d i h y d r o c o u m a r i n 1, 2 a n d 3, are given i n table I. The m a i n significant change in the a m i n o acid c o m p o s i t i o n s u b s e q u e n t to the modification is a decrease of the h i s t i d i n e c o n t e n t from 2 residues to 1.25-1 residues, depend i n g on the reagent. Methionine sulfone analyses [19] of a - c h y m o t r y p s i n i n a c t i v a t e d b y 1 or 2 have been also c a r r i e d out a n d the results shaw no significant loss of m e t h i o n i n e residue.
Those derived from the native enzyme lie n e a r n e u t r a l i t y ~ h i l e those derived from the modified enzyme migrate to~'ards the cathode, possible because of the negatively charged carboxylate group of the reagent h y d r o l y z e d d u r i n g the various treatments. Thus a n e w more acidic P a u l y negative peptide A'2 replaces A2 ~while the corresp o n d i n g P a u l y positive A'I (analogous to A1) lies n e a r the diagonal. The c o m p o s i t i o n of p e p t i d e A'2 is that expected for residues 55-64 but is devoid of h i s t i d i n e : cysteic acid 0.9 (1.0), aspartic acid 1.15 (1.0), t h r e o n i n e 1.75 (1.8), serine 1.1 (1.1), glyeine 1.35 (1.3), a l a n i n e 1.7 (1.9), valine 1.05 (1), h i s l i d i n e 0 (1), glutamic acid 0.2, leucine 0.15 (the
NATIVE
ENZYME ( . ~
MODIFIEDENZYME
9
©
~=~
O
~
- A 2 ~
c~
0
o
.............
(/ cJ
+
-t-
Fro. 6. - - Diagonal maps of peptic digests of native a-chymotrypsin and a-chymotrypsin modified by the dihydrocoumarin 3. Electrophoreses 'were performed at DH 6.5 and 3000 V for 50 minutes (in the case of the native enzyme) or one hour (in the case of the modified enzyme), in both dimensions. Cysteic acid peptides staining for histidine are hatched.
Diagonal peptide mapping. The diagonal p e p t i d e m a p p i n g method has been often used for the identification of aIkylated a m i n o acid residues i n the active site of a-chymot r y p s i n [20-2~]. Diagonal p e p t i d e maps of native ~,-chymot r y p s i n a n d a - c h y m o t r y p s i n modified by the reagent 3 are p r e s e n t e d in figure 6. They are c o m p a r a b l e to those r e p o r t e d b y B r o w n a n d Hartley [!14] and Smillie a n d Hartley [20! w i t h the exception of peptides called D3, D4. D5 by Bro~vn a n d Hartley [141, 'which ~vere not detected i n our experiments. In the diagonal map of the peptic digest of native c h y m o t r y p s i n , peptides A1 a n d A2 arise from the Cys-42 to Cys-58 d i s u l p h i d e bridge of the enzyme a n d c o n t a i n s the sequences a r o u n d His-40 (peptide A1) a n d His-57 (peptide A2). The only detectable difference b e t w e e n both diagonal maps lies in these c y s t i n e - b r i d g e d h i s t i d i n e peptides.
BIOCHIMIE, 1977, 59, n ° 3.
n u m b e r s in parentheses are those o b t a i n e d by B r o w n a n d Hartley [14] for the h i s t i d i n e peptide A2). Thus the diagonal p e p t i d e m a p p i n g method sho~'s that histidine-57 i n a-chymot r y p s i n is modified by the b i f u n c t i o n a l reagent 3. Diagonal peptide maps of a - c h y m o t r y p s i n modified by the reagent 1 w e r e also p r e p a r e d . The region ~vhere the h i s t i d i n e peptides i n the native enzyme migrated was devoid of Pauly-posifive peptides, but the n e w position of a p e p t i d e c a r r y i n g the alkylated h i s t i d i n e was not accurately established. DISCUSSION. The h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s are very efficient i n h i b i t o r s of a-chymot r y p s i n . As they are also substrates for this enzyme, their reaction ~vith ct-chymotrypsin m a y
Inactivation of a-chgmotrgpsin by ~ suicide substrates ~>. be described as a first a p p r o x i m a t i o n a c c o r d i n g to the following m i n i m a l scheme : K~ E + S ~-->
k2 ES - - - - >
k3 ES'
> E + P
L
¥ ES" in w h i c h E is the enzyme, S the reagent, P the h y d r o l y s i s p r o d u c t ; ES is the Micha~lis complex, ES' the acyl-enzyme and ES" the i n a c t i v a t e d enzyme a p p e a r i n g after f o r m a t i o n of the acylenzyme. This m e c h a n i s m seems to be the most p r o b a b l e for the following reasons : a) the i n a c t i v a t i o n reaction is not a simple him o l e c u l a r a l k y l a t i o n r e a c t i o n as s h o w n by the effects of reagent c o n c e n t r a t i o n on the rate a n d the extent of i n a c t i v a t i o n (figures 3 a n d 4). Thus, u n d e r some e x p e r i m e n t a l c o n d i t i o n s , a p a r t i a l i n a c t i v a t i o n of a - c h y m o t r y p s i n is often observed even after a long i n c u b a t i o n time ; as a matter of fact, an increase in the i n h i b i t o r c o n c e n t r a t i o n relative to the enzyme c o n c e n t r a t i o n makes the i n a c t i v a t i o n more complete. b) the i n a c t i v a t i o n reaction is p H - d e p e n d e n t w h i l e the direct a l k y l a t i o n of a - c h y m o t r y p s i n by the analogous c o u m a r i n 4 whose ester b o n d is stable, is p H - i n d e p e n d e n t [5]. The different b e h a v i o u r s of c o m p o u n d s 1 and 4 t o w a r d s o.-chyi n o t r y p s i n c a n n o t be due to the presence of differ e n t halogens because the d i h y d r o c o u m a r i n 5, the b r o m o m e t h y l a t e d analogue of 1, inactivates tt-cbym o t r y p s i n at pH 7, at the same rate as 1 a n d shows the same effect of reagent c o n c e n t r a t i o n on the extent of the reaction. This i n d e p e n d e n c e of the i n h i b i t i o n w i t h the n a t u r e of the leaving group contrasts with the usual observation for direct s u b s t i t u t i o n reactions at saturated c a r b o n atoms 'where Br- is a better leaving group t h a n C1[see for example ref. 24] a n d agrees with a mechan i s m of alkylation i m p l y i n g the i n t e r m e d i a t e form a t i o n of a q u i n o n e methide. e) T h a n k s to their reactive cis-ester b o n d a n d t h e i r good leaving group, d i h y d r o c o u m a r i n s are potential substrates for esterases a n d proteases. Tobias et al. [35] have p r e v i o u s l y s h o w n that 3 , 4 - d i h y d r o - 6 - n i t r o c o u m a r i n is a substrate for a - c h y m o t r y p s i n a n d deacylation is the rate cont r o l l i n g step in the enzymatic hydrolysis. The 3 , 4 - d i h y d r o - 6 - m e t h y l c o u m a r i n 6, w h i c h has a less good l e a v i n g group, is also h y d r o l y z e d by a-chym o t r y p s i n , but, in this case, acylation seems to be the rate l i m i t i n g step. I n d e e d the value of the k i n e t i c c o n s t a n t K m is s i m i l a r to the value of the
BIOCHIMIE, 1977, 59, n ° 3.
237
dissociation c o n s t a n t of the enzyme-substrate c o m p l e x ( d e t e r m i n e d from i n h i b i t i o n k i n e t i c m e a s u r e m e n t s as a K i v a l u e ; see results). The c o r r e s p o n d i n g h a l o m e t h y l a t e d derivative 1 reacts w i t h ct-chymoirypsin in a m a n n e r c o m p a r a b l e to 6, but the r e a c t i o n stops w h e n the enzyme has only t u r n e d over a few m o l a r equivalents of the reagent. The same respective b e h a v i o u r s of acylactivated d i h y d r o c o u m a r i n s 3 and 7 have also been observed [5]. Therefore, the rate linfiting step in the i n a c t i v a t i o n of ct-chymotrypsin by these derivatives is likely the acylation step. This last result leads to the following c o m m e n t : t h a n k s to their h y d r o p h o b i c nature, the d i h y d r o c o u m a r i n s insert easily i n the b i n d i n g pocket of ct-chymotrypsin ; their kinetic constants K m are therefore very low. Thus c o u m a r i n is also k n o w n as a good competitive i n h i b i t o r of a - c h y m o t r y p s i n [26]. However their b i n d i n g are largely u n p r o d u c tive as i n d i c a t e d by the lo~v values of koat. This b e h a v i o u r contrasts with the b e h a v i o u r of a n o t h e r sterically restricted substrate, m e t h y l d i h y d r o i s o carbostyril-3-carboxylate [27], the cyclic analogue of the specific substrate, m e t h y l N-acetyl-L-phen y l a l a n i n a t e , w h i c h scissile ester b o n d is exocyclic. The D e n a n t i o m e r of lhis c o m p o u n d (but not the L e n a n t i o m e r ) is a good substrate for a-chym o t r y p s i n (Km = 0.52.7 mM a n d kea t = 22.7 sec -1 at pH 7.9 and 25 ° ) ; its exocyclic ester group is i n d e e d correctly p o s i t i o n e d t o w a r d s the catalytic entities of the enzyme t h a n k s to the correct fit of its t w o - r i n g part in the b i n d i n g pocket of a-chym o t r y p s i n [28]. F i n a l l y all these data are c o m p a t i b l e w i t h the reaction scheme s h o w n in figure 7. After formation of the Micha~lis complex, the enzyme is acetylated on the active serine-195 ; in the t r a n s i e n t acyl-enzyme, the reactive p - h y d r o x y b e n z y l halide is t r a n s f o r m e d into q u i n o n e m e t h i d e [29] w h i c h is i n s t a n t a n e o u s l y attacked either b y a n y nucleophilie e n z y m i c residue located at p r o x i m i t y or by m e d i u m nvater. I n the first case, a chemically modified enzyme is o b t a i n e d ; in the second one, the native enzyme is restored after deacylation of the i n t e r m e d i a t e complex. The n u m b e r of molecules of radioactive dih y d r o c o u m a r i n s [14C] 1 or [3H] 3 i n c o r p o r a t e d in a - c h y m o t r y p s i n at pH 7 a n d 25 ° is slightly h i g h e r t h a n one. The a m i n o acid analyses of the modified enzyme on acid h y d r o l y s e s reveal m a i n l y a loss of 0.75-1 h i s t i d i n e residue. F r o m the data of diagonal peptide maps t h i s h i s t i d i n e has been identified as histidine-57 (at least i n tt-chymot r y p s i n i n a c t i v a t e d by 3), w h i c h is at the active site a n d whose a l k y l a t i o n is k n o w n to change tile
J.-J. Bdchet, A. D u p a i x a n d I. B l a g o e v a .
238
c a t a l y t i c p r o p e r t i e s of t h e e n z y m e . H o w e v e r o t h e r a m i n o a c i d r e s i d u e s i n ( o r n e a r to) t h e a c t i v e s i t e m i g h t b e p a r t i a l l y m o d i f i e d to a h a r d l y d e t e c t a b l e extent, as m e t h i o n i n e - 1 9 2 or tryptopban-215 w h i c h a r e s e n s i b l e to a l k y l a t i n g r e a g e n t s [30, 31].
r a t h e r r e s t r i c t i v e , at l e a s t f o r s i m p l e a l k y l a l i n g r e a g e n t s [:21], a p a r t i c u l a r r e a c t i v i t y a n d a c c e s s i b i l i t y of t h i s a m i n o a c i d r e s i d u e i n t h e i n t e r i n e diate acyl-enzyme may be responsible for this pref e r e n t i a l a t t a c k (**). TABLE II.
E+S~
Inactivation of ,~-chgmotrgpsin by various reagents at p H 7 and 25 ° .
ES~
s~, 195 acyl-enzyme
V%o. I
i o
/ N. CH 2
\ HOCH2
H o ~o
'2,,,~,~ E + P
modified enzyme
FIG. 7. - - P o s t u l a t e d m e c h a n i s m for the r e a c t i o n of a h a l o m e t h y l a t e d derivative of d i h y d r o e o u m a r i n like 1, ~with a - e h y m o t r y p s i n (see text for details). The symbol Null designates some nucleophilic enzymic residue.
Various bifunctional reagents carrying an acylating function and a second reactive chemical group have been previously used against ~-chymotrypsin. The acylating function was generally an activated ester like p-nitrophenyl ester. The second chemical group can be a normal alkylating f u n c t i o n ( l i k e - N H C O C H 2 B r ) a n d , i n t h i s case, p r e f e r e n t i a l a l k y l a t i o n of m e t h i o n i n e - 1 9 2 o c c u r s a f t e r i n i t i a l f o r m a t i o n of a n a c y l - e n z y m e [3'2, 331. T h i s s e c o n d r e a c t i v e g r o u p c a n also b e p h o t o g e n e r a t e d in situ [M, 35], a n d i n s e r t e d i n t o v a r i o u s a m i n o a c i d r e s i d u e s of t h e e n z y m e . At last, as i n o u r c a s e , i t c a n b e g e n e r a t e d a f t e r s c i s s i o n of t h e e s t e r b o n d of t h e r e a g e n t a n d f o r m a t i o n of t h e acyl-enzyme. Thus an activated benzyl halide p r e s e n t i n t h e l e a v i n g g r o u p of a q u a s i s u b s t r a t e w a s s h o ' w n to r e a c t w i t h o n e ( o r s e v e r a l ) t r y p t o p h a n r e s i d u e (s) p r o x i m a l to t h e a c t i v e s i t e of c h y m o t r y p s i n o r p a p a i n [3~, 37] (*). In this work, the activated alkylating function g e n e r a t e d i n t h e a c t i v e s i t e of a - c h y m o t r y p s i n , preferentially modifies hislidine-57. Although the r e q u i r e m e n t s f o r h i s t i d i n e a l k y l a t i o n s e e m to b e (*) Recently, a b i f u n e t i o n a l i n h i b i t o r of c~-ehymot r y p s i n able to l i b e r a t e a e a r b o n i u m ion in the active s i t e of the enzyme h a s b e e n d e s c r i b e d [,38] ; its m e c h a n i s m of action sho~ees some f e a t u r e s c o m p a r a b l e to those reported for h a l o m e t h y l a t e d derivatives of dihydrocoumarins.
BIOCHIMIE, 1977, 59, n ° 3.
Inhibitor
Diisopropyiphosphorofluoridate (DFP) (a) Phcnylmethanesulfonyl fluoride (PMSF) (a) N, N-Diphenylcarbamoyl chloride (DPCC)(b) L-(-tosylamido-2-phenyl) ethyl chloromethylketone (TPCK) (c) Benzyloxycarhonyl-L-phenylalanine bromomethylketone ( Z P B K ) (,) Z-Ala-Gly-Phe-CH~ Cl(d) Dihydrocoumarin 1 (e) Dihydrocoumarin 2 (f) Dihydrocoumarin 3 (g)
k2nd
(M-I sec-l) 45 250 480 7.7 790 100 170 >~ 20000 3O0O
(a) F r o m F a h r n e y a n d Gold [42]. (b) At pH 7,8; f r o m E r l a n g e r et al. [43]. (e) F r o m Sha~v a n d Ruseica [441. (d) At 40 ° ; f r o m M o r i h a r a a n d Oka [45]. (e) This work ; the a p p a r e n t second-order rate c o n s t a n t is det e r m i n e d i n the conditions 'where the reagent concent r a t i o n (97.5 ~M) is very m u c h larger t h a n the e n z y m e c o n c e n t r a t i o n (0.92 10~M). The r e s i d u a l activity of t h e enzyme is tested a f t e r 0.5,-2 m i n u t e s of i n c u b a t i o n time. (f) Too f a s t to be m e a s u r e d ; u - c h y m o t r y p s i n (0.5 I~M) is completely i n a c t i v a t e d a f t e r i n c u b a t i o n for one m i n u t e i n presence of 2.3 ,uM d i h y d r o e o u m a r i n 2. In a n o t h e r ikind of experiment, a - c h y m o t r y p s i n (0.009 ;~M) added at pH 7.5 and 25 ° to a med i u m c o n t a i n i n g 10 mM AcTyrOEt i n 0.5 M NaCI a n d the reagent 2 at a c o n c e n t r a t i o n as law as 0.028 aM is still completely i n h i b i t e d u n d e r one m i n u t e . (g) Approx i m a t e value at pH 6.5 ; f r o m B6chet et al. [5].
I n c o n c l u s i o n , t h e u s e of h a l o m e t h y l a t e d d e r i v a t i v e s of d i h y d r o c o u m a r i n s as i n a c t i v a t o r s of tt-chymotrypsin may be limited by the relative s o l u b i l i t y of t h e s e c o m p o u n d s , t h e i r r e l a t i v e stab i l i t y i n a q u e o u s s o l u t i o n (at p H 7 a n d 25 ° , t h e h a l f - l i v e s of i n h i b i t o r s 1 a n d 3 a r e a b o u t 65 a n d 15 m i n u t e s , r e s p e c t i v e l y ) , a n d t h e n e c e s s i t y to u s e them in a sufficient excess (except for the most s p e c i f i c d e r i v a t i v e 2). H o w e v e r t h e i r e f f i c i e n c y is h i g h . I n t a b l e II, t h e apparent s e c o n d - o r d e r r a t e c o n s t a n t s ( k o b ~ J [ I n h i b i t o r 3 ) f o r i n a c t i v a t i o n of a - c h y m o t r y p s i n b y 1, 2 a n d 3 a r e c o m p a r e d v c i t h t h e s e c o n d - o r d e r r a t e c o n s t a n t s f o r i n a c t i v a t i o n of t h i s e n z y m e b y a fe~v a c t i v e - s i t e - d i r e c t e d r e a g e n t s . It a p p e a r s t h a t h a l o m e t h y l a t e d d e r i v a t i v e s of dihydroeoumarins, e s p e c i a l l y c o m p o u n d s 2 a n d 3, (**) I n model reactions, it h a s b e e n s h o w n t h a t the d i h v d r o c o u m a r i n s 1 and 3 svere able to react w i t h imidazole in aqueous solution, yielding d e r i v a t i v e s s u b s t i t u t e d on the c a r b o n 6 [5, 39].
Inactivation
of a-chgmotrgpsin
are among the most efficient known synthetic i r r e v e r s i b l e i n h i b i t o r s of a - c h y m o t r y p s i n .
by
(< s u i c i d e
This w o r k ~vas supported by grants f r o m CNRS (Groupe de Recherches No. 13) and D6I~gation G6n6tale h la Recherche Scientifique et Technique (Conventions No. 71.7.2627 and 73.7.1687). We are very much indebted to Dr. M. W a k s e l m a n and Professor M. Vilkas for the discussions 'which led to the synthesis and enzymatic application of the h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s . We 'wish to t h a n k Dr. J. Van H e i j e n h o o r t and Mrs. A. Mantot for p e r f o r m i n g a m i n o acid analyses. We are indebted to Dr. B. Ribadeau Dumas for p e r f o r m i n g m e t h i o n i n e sulfone analyses. We also w i s h to acknowledge Dr. J. F. Petit 'who made available the use of a high-voltage electrophoresis apparatus. W e are grateful to Dr. C. Ghelis for his advices and useful discussions. We t h a n k Dr. Thusius for carefully reading the manuscript. R~su ~i~. L'c~-chymotrypsine est r a p i d e m e n t et irr~versiblem e n t inactiv~e par une s~rie de d~riv~s halom~thyl~s de d i h y d r o c o u m a r i n e s h pH 7 et 25 °. L'inactivation est d~pendante du pH, et optimale h pH neutre ; elle est aussi plus ou moins totale suivant les concentrations respectives d ' i n h i b i t e u r et d'enzyme. Ces d i h y d r o c o u m a r i n e s sont des s u b s t r a t s pour l'ct-chymotrypsine et, d u r a n t l'acte eatalytique, une f o n c t i o n alkylante latente des r~actifs est activ~e au centre actif de l'enzyme, et r~agit avec tout r6sidu nucl~ophile d'acide anmiu~ voisin. La st,0echiom6trie de la r~action des d~rives r a d i o a c t i f s c o r r e s p o n d a n t s avee 1'enzyme est l~g6rement sup~rieure h un, m a t s une h i s t i d i n e de l'ct-ehymotrypsine est surtout modifi6e. Cette histidine est identifi~e comme l'histidine-57 par la m~thode des cartes peptidiques. P a r r a p p o r t h celle d'autres r6actifs, l'efficacit~ de ces ¢ substrats-suicide >> el, en particulier, celle du d6riv6 2 p o r t e u r d'une chatne lat~rale caract~ristique des substrats de l ' a - c h y m o t r y p s i n e , est tr~s grande. REFERENCES. 1. Sha'w, E. (1970) E n z y m e s , 3rd Ed., 1, 91-146. 2. Powers, J. C..& Tuhy, P. M. (1973) Biochemistry, 12, 4767-4774. 3. Rando, R. R. (1974) Science, 185, 320-324. 4. Sigman, D. S. ,~ Mooser, G. (1975) Ann. Rev. Biochem., 44, 889-931. 5. Bdchet, J. J., Dupaix, A., Yon, J., ~ a k s e l m a n , M., Robert, J. C. & Vilkas, M. (1973) Eur. J. Biochem., 35, 527-539. 6. ~,Va:kselman, M., Hamon, J. F. ,a Vilkas, M. (1974) Tetrahedron, 30, 4069-4079. 7. Nicolle, J. P., Hamon, J. F. & W~kselman, M. (1977) Bull. Soe. Chim. Fr., in the press. 8. B~chet, J. J., Dupaix, A., Roucous, C. ~ Bonamy, A. M. (1977) Biochimie, 59, 241-246. 9. Dixon, G. H. & Neurath, H. (1957) J. Biol. Chem., 225, 1049-1059.
BIOCHIMIE, 1977, 59, n ° 3.
>>.
239
10. Wilcox, P. E., Kraut, J., Wade, R. D. & Neurath, H. (1957) Biochim. Biophys. Acta, 24, 72-78. 11. Coetzee, J. F. (1967) Progr. Phys. Org. Chem., 4, 45-92.
A c.kn olwledymen is.
substrates
12. Hirs, C. H. SV. (1967) Methods EnzymoI., 11, 59-62. 13. Spaekman, D. H., Stein, 'W. H. ~ Moore, S. (I958) Anal. Chem., 30, 1190-1206. 14. Bro~vn, J. R. ~ Hartley, B. S. (1966) Biochem. J., 101,, 214-231. 15. Heilmann, J., Barrollier, J. & Watzke, E. (1957) Hoppe-Seyler's Z. Physiol. Chem., 309. 219-220. 16. Dent, C. E. (1947) Biochem. J., 41, 240-253. 17. Gold, A. M. ,t, F a h r n e y , D. (1964) Biochemistry, 3, 783-791. 18. Gold, A. M. (1965) Biochemistry, 4, 897-901. 19. Neumann, N. P., Moore, S..& Stein, W. H. (1962) Biochemistry, l, 68-75. 20. Smi)lie, L. B. ~ Hartley, B. S. (1966) Biochem. J., 101, 232-241. 21. Stevenson, K. J. a Smillie, L. B. (1968) Can. J. Biochem., 46, 1357-1370. 22. Stevenson, K. J..& Smillie, L. B. (1970) Can. J. Biochem., 48, 364-375. 23. Jones, J. B. ~ Hysert, D. V(. (1972) Biochemistry, {11, 2726-2733. 24. Plapp, B. V. (1973) J. Biol. Chem., 248, 4896-4900. 25. Tobias, P., Heidema, J. H., Lo, K. W., Kaiser, E. T. Kezdy, F. J. (1969) J. Am. Chem. Soe., 91, 202203. 26. Wallace, R. A., Kurtz, A. N. ~ Niemann, C. (1963) Biochemistry, 2, 824-836. 27. Hein, G. E. a Niemann, C. (1962) J. Am. Chem. Soc., 84, 4487-4494. 28. Cohen, S. G., Milovanovic, A., Schultz, R. M. & Weinstein, S. Y. (1969) J. Biol. Chem., 244, 26642674. 29. Turner, A. B. (1964) Quart. Rev. (Lond.), ~8, 347360. 30. Schramm, H. J. & Lawson, W. B. (1963) HoppeSeyler's Z. Physiol. Chem., 332, 97-100. 31. Keil-Dlouha, V. ,& Keil, B. (1973) J. Mol. Biol., &l, 381-394. 32. Lawson, W. B. ,& Schramm, H. J. (1965) Biochemistry, 4, 377-386. 33. Alazard, R., B6chet, J. J., Dupaix, A. ~ Yon, J. (1973) Biochim. Biophys. Aeta, ~09, 379-396. 34. Hexter, C. S. ~ 'Weslheimer, F. H. (1971) J. Biol. Chem., 246, 3934-3938. 35. Bridges, A. J. & Kn(ywles, J. R. (1974) Biochem. J., 143, 663-668. 36. Horton, H. R. ~ Young, G. (1969) Biochim. Biophys. Aria, 194, 272-278. 37. Mole, J. E. ~ Horton, H. R. (1973) Biochemistry, 12, 5278-5285. 38. ~Vhite, E. H., Ros~xell, D. F., Politzer, I. R. & Branchini, B. R. (1975) J. Am. Chem Soc, 97, 22902291. 39. Hamon, J. F., Wakselman, M. ~ Vilkas, M. (1975) Bull. Soc. Chim. Fr., 5-6, 1387-1389. 40. Dayhoff, M. O. (1969) in Atlas of Protein Sequence and Structure, Vol. 4, National Biomedical Research F o u n d a t i o n , Silver Spring, Maryland, D 118. 41. Moore, S. (1963) J. Biol. Chem., $38, 235-237. 42. F a h r n e y , D. E. ~ Gold, A. M. (1963) J A m . Chem. Soe., 85, 997-1000. 43. Erlanger, B. F., Vratsanos, S. M., W a s s e r m a n , N. H. Cooper, A. G. (1970) Biochem. J., 1118, 421-425. 44. Sha~w, E. & Ruscica, J. (1971) Arch. Biochem. Biophys., 145, 484-489. 45. Morihara, K. ~ Oka, T. (1970) Arch. Biochem. Biophys., 138, 526-531.
BIOCHIMIE, 1977, 59, 231-239.
Inactivation of -chymotrypsin by new bifunctional reagents halomethylated derivatives of dihydrocoumarins. Jean-Jacques
B~CHET ~ , A l a i n DUPAIX a n d I v a BLAGOEVA ~.
Laboratoire d ' E n z y m o l o g i e P h y s i c o - c h i m i q u e et Mol~culaire, Groupe de R e c h e r c h e s du CNRS associ~ it rUniversit~ de Paris-Sad, Orsay, 91~05, France. (29-10-1976). Summary. - - ~ - C h y m o t r y p s i n is r a p i d l y a n d completely i n a c t i v a t e d by a series of h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s at pH 7 a n d 25 °. The i n a c t i v a t i o n is p H - d e p e n d e n t a n d o p t i m a l at n e u t r a l pH ; it is also more or less complete depending on the excess of i n h i b i t o r w i t h respect to t h e enzyme. These compounds are s u b s t r a t e s for ~ - c h y m o t r y p s i n and, d u r i n g the c a t a l y t i c process, a l a t e n t a l k y l a t i n g f u n c t i o n of the reagent is activated at the active site of the e n z y m e a n d reacts w i t h some vicinal nueleophilic a m i n o acid residues. The s t o i c h i o m e t r y of the r e a c t i o n of the corresponding radioactive reagents w i t h the e n z y m e is slightly superior to one, b u t one h i s t i d i n e residue of a - e h y m o t r y p s i n is m a i n l y modified. This h i s t i d i n e is identified as histidine-57 b y the d i a g o n a l peptide m a p p i n g method. In c o m p a r i s o n 'with o t h e r reagents, the efficiency of these ¢ suicide s u b s t r a t e s ~ a n d p a r t i c u l a r l y t h a t one of a derivative (compound 2) c a r r y i n g a s u b s t r a t e - l i k e side e h a i n is f o u n d to be very high.
Tile s e l e c t i v e m o d i f i c a t i o n of a m i n o a c i d s p r e s e n t i n t h e a c t i v e s i t e s of e n z y m e s is o f t e n o b t a i n e d b y t h e u s e of a c t i v e - s i t e - d i r e c t e d r e a g e n t s [1]. These reagents have substrate-like structural f e a t u r e s ' w h i c h p r o m o t e t h e f o r m a t i o n of a n adsorption complex with the enzyme ; a chemical g r o u p of t h e r e a g e n t m a y t h e n a c h i e v e a c o v a l e n t m o d i f i c a t i o n w i t h i n t h e a c t i v e site. T h u s p r o t e a s e s may be specifically alkylated by peptide chlorom e t h y l k e t o n e s [2, a n d r e f e r e n c e s t h e r e i n ] . The use of substrates carrying a latent chemical g r o u p a c t i v a b l e i n t h e a c t i v e s i t e of a n e n z y m e d u r i n g t h e c a t a l y t i c p r o c e s s , m a y also l e a d to s o m e i n f o r m a t i o n o n t h e n a t u r e of d i f f e r e n t a m i n o acid residues present in this active site and their respective accessibility and reactivity. This kind of inhibitor has been recently reviewed by Rando [3] a n d S i g m a n a n d M o o s e r [4] a n d h a s b e e n def i n e d as <
T h e h a l o m e t h y l a t e d d e r i v a t i v e s of d i h y d r o c o u m a r i n s ( l i k e c o m p o u n d 1 i n f i g u r e 1) a r e p o t e n tially such bifunctional reagents for esterases and proteases having an esterolytic activity. These compounds possess a reactive ester bond ~-ith a cis c o n f i g u r a t i o n , a n d a n a I k y l a t i n g f u n c t i o n
XCH2
1
1 ~ X=CI 5,
Br
X CH2
Br
3,
Abbreviations used are : CT, (x-chymotrypsin ; AcTyrOEt, N-acetyl-L-tyrosine ethyl e s t e r ; Mes, 2-(Nm o r p h o l i ~ o ) e t h a n e s u l f o n i c acid ; Hepes, N-2-hydroxye t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid. Enzymes : c h y m o t r y p s i n (EC 3.4.4.5) ; pepsin (EC 3.4.4.1). (*) This a p p e l l a t i o n of <
X=Br
6 ~X=H
X=B,
BrCH2
4
7~X=H FIG.
1. --
Structure of compounds.
w h i c h a p p e a r s a f t e r h y d r o l y t i c c l e a v a g e of t h e ester bond. Indeed the appearing p-hydroxybenzyl h a l i d e is a c t i v a t e d i n t o q u i n o n e m e t h i d e w h i c h reacts very rapidly with any neighboring nuc l e o p h i l i c r e s i d u e [5, a n d r e f e r e n c e s t h e r e i n ] . 18
J.-J. Bdchet, A. Dupaix and I. Blagoeua.
232
It has been p r e v i o u s l y s h o w n that 3,4-dih y d r o 3 , 4 - d i b r o m o - 6 - b r o m o m e t h y l c o u m a r i n (comp o u n d 3), thanks to these features, q u i c k l y inactivated (~-chymotrypsin ES]. H o w e v e r this d i h y d r o c o u m a r i n was r a t h e r u n s t a b l e i n aqueous solution because of a s p o n t a n e o u s d e h y d r o h a l o g e n a t i o n r e a c t i o n on its c a r b o n s 3 a n d 4. Therefore the more simple d i h y d r o c o u m a r i n 1 a n d its analogue 2 c a r r y i n g a substrate-like b e n z y l side c h a i n have n o w been p r e p a r e d b y W a k s e l m a n el al. E6, 7] i n the L a b o r a t o i r e de Chimie Organique Biologique d'Orsay. We study in this p a p e r t h e i r effects u p o n (t-chymotrypsin a n d c o m p a r e them to those of the acyl-activated d i h y d r o c o u m a r i n 3. I n the accomp a n y i n g p a p e r E8~, the action of these reagents t o w a r d s various proteases a n d esterases is reported. EXPERIMENTAL PROCEDURE. MATERIALS.
a - C h y m o t r y p s i n was a salt-free, three-times crystallized p r o d u c t p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l Corp. It was dissolved i n 0.001 N HC1 a n d kept at 4 ° before use. Its c o n c e n t r a t i o n in solution was s p e c t r o p h o t o m e t r i c a l l y d e t e r m i n e d at 280 n m u s i n g a m o l a r e x t i n c t i o n coefficient of 5 × 104 [9] a n d a m o l e c u l a r weight of 2.5 × 104 [10]. P e p s i n was a twice-crystallized p r o d u c t , purchased from W o r t h i n g t o n Biochemical Corp. N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) was o b t a i n e d from Mann Research Laboratories. The buffers used w e r e N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid (Hepes)-NaOH a n d 2 - ( N - m o r p h o l i n o ) - e t h a n e s u l f o n i c acid (Mes)-NaOH whose sulfonic acids w e r e p u r c h a s e d from Calbiochem Chemical Co. Acetate, borate a n d phosphate buffers w e r e p r e p a r e d from P r o l a b o products. Acetonitrile w a s purified a c c o r d i n g to Coetzee !11]. PREPARATION AND CHARACTERISATION OF DIHYDROCOUMARINS AND COUMARINS.
Most of the c o m p o u n d s w e r e synthesized a n d c h a r a c t e r i z e d as r e p o r t e d p r e v i o u s l y [5, 6~. The d i h y d r o c o u m a r i n s 2 a n d 5 have been r e c e n t l y p r e p a r e d ET]. Some special care was taken d u r i n g the use of the d i h y d r o c o u m a r i n 2 w h i c h m a y be toxic to h u m a n s because of possible a n t i c o a g u l a n t p r o p e r t i e s E7]. The syntheses of the r a d i o a c t i v e d i h y d r o c o u m a r i n s [14CJ 1 a n d [~H] 3 have been d e s c r i b e d b y W a k s e l m a n el al. [6]. The specific r a d i o a c i i vities of [I4C] 1 and IaH] 3 c a r r y i n g the radio-
BIOCHIMIE, 1977, 59, n ° 3.
active atoms on the c h l o r o m e t h y l group was 0.5 I~Ci/mM a n d 730 ~Ci/mM, respectively. The r a d i o a c t i v e derivative EI~C] 2 could not be prepared. The ultra-violet a b s o r p t i o n spectra of the differ e n t d i h y d r o c o u m a r i n s in e t h a n o l w e r e r e c o r d e d before each use of the c o m p o u n d s ; they w e r e characterized by small peaks b e t w e e n 290 n m a n d 260 n m and a strong increase of the a b s o r b a n c e between 200 n m and 240 n m : for 1, ~ma~ (EtOH) 270 n m (e 750 M-1 cm -1) a n d 279 n m (e 610 M-1 cm -1) ; for 2, )~n~ax(EtOH) 265 n m (e 950 M-1 cm-1), 268 n m (e 910 M-1 cm-1) a n d 280 n m (e 570 M-1 cm -1) ; for 3, k (EtOH) 285 n m (shoulder, 1900 M-1 cm-1) ; for 5, kmax (EtOH) 272 n m (~ 990 M-1 cm-1) a n d 280 n m (e 890 M-1 cm-1) ; for 6, k.... (EtOH) 280 n m (e 925 M-1 cm -1) a n d 272 n m (e 1010 M-1 cm-I) ; for 7, )~ (EtOH) 290 n m (shoulder, ~ 2~080 M-~ c m l i n s t e a d of 2700 M~1 cin-L at 287 nm, as p r e v i o u s l y r e p o r t e d [5] for a p r o d u c t slightly decomposed). METHODS.
Reactions of the dihgdrocoumarins with ¢x-chgmotrgpsin. 10-100 i~l of a freshly p r e p a r e d solution of 10 mM d i h y d r o c o u m a r i n in acetonitrile were added w i t h s w i r l i n g to a solution of (t-chymot r y p s i n (at final c o n c e n t r a t i o n s 4-30 ,~M) in 5 ml of 25 mM Hepes-NaOH or Mes-NaOH buffers (0.5 M NaC1 ; final p e r c e n t of acetonitrile, 0.2-2), at a given pH a n d 25 °. After different i n c u b a t i o n periods w i t h i n one hour, the c h y m o t r y p t i c activity of a small aliquot w a s tested against AcTyrOEt (10 mM or 5 raM) i n 10 ml of 0.1 M NaC1 at pH 7.5 a n d 25 °. The e n z y m a t i c h y d r o l y s i s of AcTyrOEt was followed b y pH star m e a s u r e m e n t s u s i n g a Vibron p H m e t e r model 33 B (Electronic I n s t r u m e n t s Ltd). In the same e x p e r i m e n t a l c o n d i t i o n s a n d in the absence of reagent, it was verified that the enzyme did not s p o n t a n e o u s l y lose significant activity.
Spontaneous and enzymatic hgdrolgses of dihgdrocoumarins. Kinetic m e a s u r e m e n t s w e r e c a r r i e d out at 25 ° in aqueous solutions (0.5 M NaC1) c o n t a i n i n g 1 p e r cent (v/v) acetonitrile. E m p l o y e d buffers were 0.025 M Mes-NaOH b e t w e e n pH 6 and 7 ; 0.025 M Hepes-NaOH b e t w e e n p H 7 a n d 8 ; 0.05 M b o r i c a c i d - b o r a x b e t w e e n pH 8 a n d 9 ; 0.0125 M boraxNaOH b e t w e e n pH 9 a n d 10. The s p o n t a n e o u s a n d enzymatic h y d r o l y s e s of the d i h y d r o c o u m a r i n s 1 a n d 6 w e r e followed b y s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s w i t h a Cary 16
Inactivation of a-chymotrypsin by <
Preparation of the inactivated ¢t-chymotrypsin. c¢-Chymotrypsin (0.4-0.6 m g / m l ) was i n c u b a t e d in the p r e s e n c e of a large excess of reagent ([1] = 0.5 mM or [21 ~ 0.07 mM or [3] ~ 0.2 raM), in a given volume (5 ml-250 ml) of 0.025 M HepesNaOH buffer at pH 7 a n d 25 ° for 30 m i n u t e s (with the reagent 1) or 15 m i n u t e s (with the reagents 2 or 3). The e n z y m a t i c activity was m e a s u r e d before a n d after i n c u b a t i o n . Then, the pH value of the solution w a s adjusted to pH 3 ~with HC1 or formic acid (not in the case of the enzyme i n a c t i v a t e d by 3). The modified ¢t-chymotrypsin ~ a s either filtered t h r o u g h Sephadex G 25 e q u i l i b r a t e d w i t h 0.001 N HC1 at 4 °, or exhaustively dialyzed w i t h twice-boiled dialysis bags against 0.001 N HC1 at 4 ° d u r i n g 48 or 72 h o u r s if large volumes of solu-
BIOCHIMIE, 1977, 59, n ° 3.
233
tion were nsed. I n this last case, the modified enzyme was ultrafiltrated a n d c o n c e n t r a t e d on Diaflo PM-10 or UM-10 m e m b r a n e (Amicon Corp.). F i n a l l y the enzyme solution was passed t h r o u g h a 0.22 .!xin Millipore filter and stored at 4 °. (t-Chymotrypsin modified by the r a d i o a c t i v e derivative E14C] 1 was p r e p a r e d in the same m a n n e r as described above and its r a d i o a c t i v i t y was d e t e r m i n e d as p r e v i o u s l y reported ES].
Amino acid analyses. Native or modified a - c h y m o t r y p s i n was at first filtered t h r o u g h Sephadex G-25 e q u i l i b r a t e d w i t h 0.001 N HCI. Samples of p r o t e i n (1-1.5 rag) were then lyophilized and, occasionally, p e r f o r m i c acid oxidized [12] a n d lyophilized again. T h e y ~-ere m i x e d w i t h c o n s t a n t - b o i l i n g HC1, deaerated a n d h y d r o l y z e d in vacuo for 22 hours at 110 °. Hydrolysates were evaporated to d r y n e s s and the residues taken up i n 0.2 M s o d i u m citrate buffer pH 2.2. Amino acid analyses were p e r f o r m e d by the methgd of S p a c k m a n el al. I13] with a Beckm a n Multichrom automatic a m i n o acid a n a l y s e r coupled with an I n f o t r o n i c s model C.R.S. 210 integrator. Molar ratios of a m i n o acids were calculated on the basis of l e u c i n e ---- 19.
Diagonal paper electrophoresis. Diagonal p a p e r electrophoretograms of native or i n h i b i t e d c h y m o t r y p s i n s were p r e p a r e d accord i n g to the method of B r o w n a n d Hartley [14]. T y p i c a l l y , the native or modified c h y m o t r y p s i n (50 rag) was digested w i t h p e p s i n (5 mg) i n 10 ml of 5 per cent formic acid for 18 hours at 37 °. The peptic digest was spotted (1 m g / c m ) as a 3-cm wide b a n d in the center of a sheet of W h a t m a n 3MM paper. It was then subjected to high-voltage electrophoresis p e r f o r m e d w i t h a Gilson F r a n c e model D a p p a r a t u s u s i n g varsol coolant a n d a buffer of adequate pH, at 3000 V at pH 6.5 for 1 h o u r (or 50 rain in the first e x p e r i m e n t s w i t h the native enzyme). A 3-cm w i d e strip was cut from the edge of the dried sheet and exposed for 2 h o u r s at room t e m p e r a t u r e in the vapors of p e r f o r m i c acid. After drying, the oxidized strip was sewn across the center of a n o t h e r sheet of W h a t m a n 3MM p a p e r and subjected again to highvoltage electrophoresis at pH 6.5 at 3000 V for 1 hour. The peptides were detected with the cadm i u m - n i n h y d r i n reagent [15] a n d those contain i n g h i s t i d i n e w i t h the P a u l y reagent [16]. After e x a m i n a t i o n of the o b t a i n e d d i a g o n a l p e p t i d e map, the p u r i f i c a t i o n of p a r t i c u l a r cysteic acid peptides called A'I a n d A'2 (see results) was made in the following way. The cystine p e p t i d e p a r e n t of A'I a n d A'2 was cut out as a b a n d from
J.-J. B d c h e t , A . D u p a i x a n d I. B l a g o e v a .
234
the first electrophoretogram and oxidized for 2 h o u r s at r o o m t e m p e r a t u r e i n t h e v a p o r s of p e r fornfic acid. After drying, the oxidized strip was s e w n o n to a s e c o n d s h e e t of W h a t m a n 3MM paper and the peptides were then purified by h i g h - v o l t a g e e l e c t r o p h o r e s i s a t 4500 V a t p H 6.5 f o r 45 m i n u t e s . C y s t e i c a c i d p e p t i d e s ' w e r e d e t e c ted on side strips with the cadmium-ninhydrin r e a g e n t a n d e l u t e d w i t h 0.1 M NH~. A f t e r e v a p o r a t i o n to d r y n e s s , p e p t i d e s w e r e h y d r o l y z e d i n 5.7 N HC1 i n s e a l e d e v a c u a t e d t u b e s f o r 22 h o u r s at 110 ° f o r a m i n o a c i d a n a l y s e s .
i n f i g u r e 4, a s l i g h t e x c e s s of a b o u t 2-3 t i m e s of t h e r e a g e n t 2 o n t h e e n z y m e is s u f f i c i e n t to i n a c t i vate a-chymotrypsin completely, while an excess of 20-30 t i m e s of t h e r e a g e n t 1 o n t h e e n z y m e is r e q u i r e d to o b t a i n t h e s a m e r e s u l t .
10(
~
CJ
t~75
~
[_0
[
' '
19,65.M
>-
J = 35 . M
RESULTS.
<
E f f e c t of p H on the inactivation of ~J.-chymotrypsin by h a l o m e t h y l a t e d derivatives of dihydrocoumarins. A t n e u t r a l p H , t h e d i h y d r o c o u m a r i n s 1, 2 a n d 3 ( i n e x c e s s of 10-40 t i m e s o n t h e e n z y m e ) i n a c t i vate completely and irreversibly a-ehymotrypsin within a few minutes. However, at more aeidic p H v a l u e s , t h e e n z y m e is less c o m p l e t e l y a n d q u i c k l y i n a c t i v a t e d (figure 2). T h i s s a m e p h e n o m e n o n is o b s e r v e d f o r all t h r e e r e a g e n t s .
i2~
I :
0
=
~
A"
[
10
20
.
.
=,
30 40 TIME ffmnl
z 75
pH 5.55
•
pH 6.05
'
-
•
0
O
10
20
30 40 TIME I m m )
50
i
i
60
70
FIG. 2. - - Effect of pH on the inhibition of a-chymotrypsin by 3,$-dihydro-6-chloromethylcoumarin 1 at 25°: (©) pH 5.55 ;(O) pH 6.05; (El) pH 6.45; (n) pH 7.05 ; (A) pH 7.45; (A) pH 8.3. The enzyme concent r a t i o n 'was 4.95 uM ; the reagent c o n c e n t r a t i o n was 96 ittM i n 0.025 M Hepes-NaOH or Mes-NaOH buffers (0.5 M NaC1 ; 1 per cent (v/v) acetonitrile).
of d i h g d r o c o u m a r i n s .
E f f e c t of reagent concentration on the extent of inactivation. At a g i v e n p H v a l u e , t h e r e s i d u a l a c t i v i t y o b s e r v e d a t t h e e n d of t h e r e a c t i o n d e p e n d s o n t h e m o l a r r a t i o of i n h i b i t o r to e n z y m e a n d a n a d e q u a t e e x c e s s of i n h i b i t o r is n e c e s s a r y to p r o d u c e a c o m p l e t e i n a c t i v a t i o n ( f i g u r e 3). Moreover this phenomenon depends on the s t r u c t u r e of t h e d i h y d r o c o u m a r i n . T h u s as s h o w n
0
75
'/ 2
3
1
~_50 z
--25
BIOCHIMIE, 1977, 59, n ° 3.
7=0
-
100
pH 7,05
m
6=0
In fact, dihydrocoumarins are substrates for a - c h y m o t r y p s i n . T h u s , t h e k i n e t i c c o n s t a n t s K m, kca t a n d k,.at/K m f o r t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of t h e s i m p l e s t 3 , 4 - d i h y d r o - 6 - m e t h y l coumarin 6 ('which does not irreversibly inactiv a t e t h e e n z y m e ) a r e e q u a l to 0.22 mM, 0.021 s e c -I a n d 95 M q s e c -1 r e s p e c t i v e l y , at p H 7.5 a n d 25 °.
0
50
510
FIG. 3. Effect of reagent concentration on the percentage inhibition of ~-chymolrypsin by 3,$-dihydro-6-chloromethylcoumarin 1 at pH 7.05 and '25°. The e n z y m e c o n c e n t r a t i o n w a s 4.95 IvM a n d the concent r a t i o n of the i n h i b i t o r I 'was v a r i a b l e : (©) no reagent ; (O) 19.65 ~tM ; ([3) 35 ~tM ; (ll) 50 ~M ; (~) 95 I~M ; (&) 195 uM.
E n z g m a t i c hgdrolgsis 00
50 . M
La
~
O0
•
5
10 [INHIBITOR]/IENZYMEi
15
'~0
Fro. 4. - - Percentage inhibition as a function of the molar ratio of dihydrocoumarine 1,2 or 3 Iwith respect to u-chymotrypsin at pH 7 and 25 ° : (0), c o n c e n t r a t i o n of the reagent I = 19.3 aM-196 UM, c o n c e n t r a t i o n of the e n z y m e = 4.93 lu.M, i n c u b a t i o n t i m e = one h o u r ; (@), c o n c e n t r a t i o n of the r e a g e n t 2 : 5.87 ,~M - 68.2 ~M, e n z y m e c o n c e n t r a t i o n = 10.9 ~M, i n c u b a t i o n t i m e 15 m i n u t e s ; (A), c o n c e n t r a t i o n of the reagent 3 ----24 i~M- 185 ,~tM, enzyme c o n c e n t r a t i o n = 25 ~tM, incub a t i o n t i m e ---- 15 minutes.
Inactivation of a - c h g m o t r g p s i n Furthermore, this titive i n h i b i t o r in specific substrate constant Ki equal Km value.
l a c t o n e 6 b e h a v e s as a c o m p e the e n z y m a t i c h y d r o l y s i s of the AcTyrOEt, with an inhibition to 0.2 mM a n d i d e n t i c a l to t h e
I n c a s e of t h e m o r e s p e c i f i c r e a c t i o n w i t h t h e hindered dihydrocoumarin 2, ~ - e h y m o t r y p s i n turns over between one and two molar equivalents o f t h e c o m p o u n d 2, b e f o r e it b e c o m e s c o m p l e t e l y i n a c t i v a t e d (figure 4 ) ; t h e l o w s o l u b i l i t y of
T h e d i h y d r o c o n m a r i n 1 is also h y d r o l y z e d b y a - c h y m o t r y p s i n (*) at a r a t e s o m e w h a t h i g h e r t h a n 6, b u t t h e e n z y m a t i c r e a c t i o n b e c o m e s n e g l i gible in a few m i n u t e s b e f o r e c o m p l e t e h y d r o l y s i s o f t h e s u b s t r a t e , as s h o w n i n figure 5. F o r t h e r e a c t i o n of 1 (in a s u f f i c i e n t e x c e s s ) w i t h ¢t-chym o t r y p s i n at p H 7.5 a n d 25 ° , t h e e n z y m e t u r n s over about 6 m o l a r e q u i v a l e n t s of 1 b e f o r e complete inactivation. For comparison, the slower and more continuous trypsin-catalyzed hydrolysis of this d i h y d r o c o u m a r i n is also s h o w n in f i g u r e 5 ; as r e p o r t e d i n t h e a c c o m p a n y i n g p a p e r [8], t h e i n a c t i v a t i o n o f t r y p s i n b y 1 is r e l a tively slow and uncomplete. (% It has been verified t h a t the rates of the spontaneous h y d r o l y s e s of the d i h y d r o c o u m a r i n s 1 and 6 were relatively negligible 'with regard to those of their enzymatic hydrolyses, at pH 7.5 and 25 ° . In the alkaline pH range 7.5-9.5, the second-order rate constants ko~- for the alkaline hydrolyses of compounds 1 and 6 are 310 M-1 see-1 and 170 M-1 sec-L respectively. Between pH 6 and 8, some w a t e r - c a t a lyzed hydrolysis of compound 1 is observed ~vith a first-order rate c o n s t a n t ko equal to 1.5 X 10-4 sec-1 (spectrophotometric m e a s u r e m e n t s at 250 rim).
235
by ¢ suicide s u b s t r a t e s >~.
i,'.. °"°"°*°°
E =0
,3 E : 29.5~M
TIME (mm)
FIG. 5. - - K i n e t i c runs of the a-chymotrypsin-catalyzed hydrolysis of dihydrocoumarin 1 at pH 7.5 and 25°; spectrophotometric m e a s u r e m e n t s ; reagent conc e n t r a t i o n = 196 j~M ; variable enzyme concentration, E. A kinetic run of the t r y p s i n - c a t a l y z e d h y d r o l y s i s of d i h y d r o c o u m a r i n 1 at pH 7.5 and 25" is also sho~vn ( @ Q O O ) : reagent concentration = 196 uM ; enzyme c o n c e n t r a t i o n = 14.1 ~M. The enzyme w a s p r e s e n t in both sample and reference cells. The final absorbance of the h y d r o l y s i s product of 1 is also given (. . . . . ).
TABLE I.
Amino acid composition of native a-chymotrypsin and a-chymotrypsin modified by halomethylated derivatives of dihydrocoumarins (a). Native C T Amino acid
I
Theoretical values (b)
Experimental values
C T modified (3 T modified by 2 by I
C T modilied by 3 (c }
I Aspartic acid . . . . . . . . . Threonine (d) . . . . . . . . Serine (d) . . . . . . . . . . . Glutamic acid . . . . . . . Proline . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . Half cystine (e) . . . . . ¥aline . . . . . . . . . . . . . . . Methionine . . . . . . . . . . .
22 22 27 15 9 23 22 10 23 2
Isoleucine . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . Phenylalanine . . . . . . . . Lysine . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . Arginine . . . . . . . . . . . . .
10 19 4 6 14 2 3
22 18.3 185 15.3 89 23.3 22.2 10.2 22.75 1.95 (f) 1.6(g) 9.4 19 3.9 5.7 14.3 1.95 3.05
22.4 18.2 18.2 15.45 9.2 24.1 22.6 10 23.45 1.95 (I) 1.45 (g) 9.45 19 4 5.9 13.9 1,25 2.9
22.7 18.55 18.65 15.3 9.0 23.1 22.1 10 22.85 2.2 (f) 1.65 (g) 9.4 19 3.9 5.8 13.85 1.1 2.95
21.3 21.3 24.8 16.5 9.8 23.2 22.5 21 2 9.7 19 3.3 64 14.2 1.0 3.1
(a) The m e a s u r e d values are the average of five analyses. (b) F r o m Dayhoff [40]. (c) F r o m B6chet et al. [15]. (d) Not corrected for h y d r o l y t i c destruction. (e) Determined as eysteic acid a f t e r p e r f o r m i c o x y d a t i o n and corrected for 94 per cent recovery [4,1]. (f) Sum of m e t h i o n i n e and m e t h i o n i n e sulfoxides. (g) Determined as m e t h i o n i n e sulfone after performic oxydation and not corrected for o v e r o x y d a t i o n [41].
BIOCHIMIE, 1977, 59, n ° 3.
236
J.-J. Bdchet, A. D u p a i x and I. Bla~!oeva.
the reagent did not h o w e v e r measurement.
allo'w its direct
Sloiehiometry of the inactivation reaction. a - C h y i u o t r y p s i n i n c u b a t e d i n the p r e s e n c e of the d i h y d r o c o u m a r i n s 1, 2 a n d 3 i n a large excess at pH 7 is i r r e v e r s i b l y i n a c t i v a t e d a n d does not recover significant activity after dialysis a n d t r e a t m e n t w i t h 1 M h y d r o x y l a m i n e at pH 7.5 a n d 20 ° for 24 hours. I n c u b a t i o n e x p e r i m e n t s ~¢ith radioactive derivatives [14CJ 1 a n d [3H] 3 [ref. 5] in excess at pH 7 and 25 ° show that 1.3 _+ 0.1 a n d 1.25 _+ 0.1 molecules of the labeled reagent are i n c o r p o rated per molecule of protein. W h e n the enzymes has been p r e v i o u s l y completely i n h i b i t e d b y p h e n y h n e t h a n e s u l f o n y l fluoride w h i c h reacts w i t h the active serine 195 p17, 18], a negligible i n c o r p o r a t i o n of the reagent [14C] 1 i n the p r o t e i n (about 0.08 molecules of reagent per molecule of p r o t e i n ) is observed).
Amino acid analyses of the inactivated enzyme. A m i n o acid analyses of a - c h y m o t r y p s i n , before and after i n a c t i v a t i o n by the d i h y d r o c o u m a r i n 1, 2 a n d 3, are given i n table I. The m a i n significant change in the a m i n o acid c o m p o s i t i o n s u b s e q u e n t to the modification is a decrease of the h i s t i d i n e c o n t e n t from 2 residues to 1.25-1 residues, depend i n g on the reagent. Methionine sulfone analyses [19] of a - c h y m o t r y p s i n i n a c t i v a t e d b y 1 or 2 have been also c a r r i e d out a n d the results shaw no significant loss of m e t h i o n i n e residue.
Those derived from the native enzyme lie n e a r n e u t r a l i t y ~ h i l e those derived from the modified enzyme migrate to~'ards the cathode, possible because of the negatively charged carboxylate group of the reagent h y d r o l y z e d d u r i n g the various treatments. Thus a n e w more acidic P a u l y negative peptide A'2 replaces A2 ~while the corresp o n d i n g P a u l y positive A'I (analogous to A1) lies n e a r the diagonal. The c o m p o s i t i o n of p e p t i d e A'2 is that expected for residues 55-64 but is devoid of h i s t i d i n e : cysteic acid 0.9 (1.0), aspartic acid 1.15 (1.0), t h r e o n i n e 1.75 (1.8), serine 1.1 (1.1), glyeine 1.35 (1.3), a l a n i n e 1.7 (1.9), valine 1.05 (1), h i s l i d i n e 0 (1), glutamic acid 0.2, leucine 0.15 (the
NATIVE
ENZYME ( . ~
MODIFIEDENZYME
9
©
~=~
O
~
- A 2 ~
c~
0
o
.............
(/ cJ
+
-t-
Fro. 6. - - Diagonal maps of peptic digests of native a-chymotrypsin and a-chymotrypsin modified by the dihydrocoumarin 3. Electrophoreses 'were performed at DH 6.5 and 3000 V for 50 minutes (in the case of the native enzyme) or one hour (in the case of the modified enzyme), in both dimensions. Cysteic acid peptides staining for histidine are hatched.
Diagonal peptide mapping. The diagonal p e p t i d e m a p p i n g method has been often used for the identification of aIkylated a m i n o acid residues i n the active site of a-chymot r y p s i n [20-2~]. Diagonal p e p t i d e maps of native ~,-chymot r y p s i n a n d a - c h y m o t r y p s i n modified by the reagent 3 are p r e s e n t e d in figure 6. They are c o m p a r a b l e to those r e p o r t e d b y B r o w n a n d Hartley [!14] and Smillie a n d Hartley [20! w i t h the exception of peptides called D3, D4. D5 by Bro~vn a n d Hartley [141, 'which ~vere not detected i n our experiments. In the diagonal map of the peptic digest of native c h y m o t r y p s i n , peptides A1 a n d A2 arise from the Cys-42 to Cys-58 d i s u l p h i d e bridge of the enzyme a n d c o n t a i n s the sequences a r o u n d His-40 (peptide A1) a n d His-57 (peptide A2). The only detectable difference b e t w e e n both diagonal maps lies in these c y s t i n e - b r i d g e d h i s t i d i n e peptides.
BIOCHIMIE, 1977, 59, n ° 3.
n u m b e r s in parentheses are those o b t a i n e d by B r o w n a n d Hartley [14] for the h i s t i d i n e peptide A2). Thus the diagonal p e p t i d e m a p p i n g method sho~'s that histidine-57 i n a-chymot r y p s i n is modified by the b i f u n c t i o n a l reagent 3. Diagonal peptide maps of a - c h y m o t r y p s i n modified by the reagent 1 w e r e also p r e p a r e d . The region ~vhere the h i s t i d i n e peptides i n the native enzyme migrated was devoid of Pauly-posifive peptides, but the n e w position of a p e p t i d e c a r r y i n g the alkylated h i s t i d i n e was not accurately established. DISCUSSION. The h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s are very efficient i n h i b i t o r s of a-chymot r y p s i n . As they are also substrates for this enzyme, their reaction ~vith ct-chymotrypsin m a y
Inactivation of a-chgmotrgpsin by ~ suicide substrates ~>. be described as a first a p p r o x i m a t i o n a c c o r d i n g to the following m i n i m a l scheme : K~ E + S ~-->
k2 ES - - - - >
k3 ES'
> E + P
L
¥ ES" in w h i c h E is the enzyme, S the reagent, P the h y d r o l y s i s p r o d u c t ; ES is the Micha~lis complex, ES' the acyl-enzyme and ES" the i n a c t i v a t e d enzyme a p p e a r i n g after f o r m a t i o n of the acylenzyme. This m e c h a n i s m seems to be the most p r o b a b l e for the following reasons : a) the i n a c t i v a t i o n reaction is not a simple him o l e c u l a r a l k y l a t i o n r e a c t i o n as s h o w n by the effects of reagent c o n c e n t r a t i o n on the rate a n d the extent of i n a c t i v a t i o n (figures 3 a n d 4). Thus, u n d e r some e x p e r i m e n t a l c o n d i t i o n s , a p a r t i a l i n a c t i v a t i o n of a - c h y m o t r y p s i n is often observed even after a long i n c u b a t i o n time ; as a matter of fact, an increase in the i n h i b i t o r c o n c e n t r a t i o n relative to the enzyme c o n c e n t r a t i o n makes the i n a c t i v a t i o n more complete. b) the i n a c t i v a t i o n reaction is p H - d e p e n d e n t w h i l e the direct a l k y l a t i o n of a - c h y m o t r y p s i n by the analogous c o u m a r i n 4 whose ester b o n d is stable, is p H - i n d e p e n d e n t [5]. The different b e h a v i o u r s of c o m p o u n d s 1 and 4 t o w a r d s o.-chyi n o t r y p s i n c a n n o t be due to the presence of differ e n t halogens because the d i h y d r o c o u m a r i n 5, the b r o m o m e t h y l a t e d analogue of 1, inactivates tt-cbym o t r y p s i n at pH 7, at the same rate as 1 a n d shows the same effect of reagent c o n c e n t r a t i o n on the extent of the reaction. This i n d e p e n d e n c e of the i n h i b i t i o n w i t h the n a t u r e of the leaving group contrasts with the usual observation for direct s u b s t i t u t i o n reactions at saturated c a r b o n atoms 'where Br- is a better leaving group t h a n C1[see for example ref. 24] a n d agrees with a mechan i s m of alkylation i m p l y i n g the i n t e r m e d i a t e form a t i o n of a q u i n o n e methide. e) T h a n k s to their reactive cis-ester b o n d a n d t h e i r good leaving group, d i h y d r o c o u m a r i n s are potential substrates for esterases a n d proteases. Tobias et al. [35] have p r e v i o u s l y s h o w n that 3 , 4 - d i h y d r o - 6 - n i t r o c o u m a r i n is a substrate for a - c h y m o t r y p s i n a n d deacylation is the rate cont r o l l i n g step in the enzymatic hydrolysis. The 3 , 4 - d i h y d r o - 6 - m e t h y l c o u m a r i n 6, w h i c h has a less good l e a v i n g group, is also h y d r o l y z e d by a-chym o t r y p s i n , but, in this case, acylation seems to be the rate l i m i t i n g step. I n d e e d the value of the k i n e t i c c o n s t a n t K m is s i m i l a r to the value of the
BIOCHIMIE, 1977, 59, n ° 3.
237
dissociation c o n s t a n t of the enzyme-substrate c o m p l e x ( d e t e r m i n e d from i n h i b i t i o n k i n e t i c m e a s u r e m e n t s as a K i v a l u e ; see results). The c o r r e s p o n d i n g h a l o m e t h y l a t e d derivative 1 reacts w i t h ct-chymoirypsin in a m a n n e r c o m p a r a b l e to 6, but the r e a c t i o n stops w h e n the enzyme has only t u r n e d over a few m o l a r equivalents of the reagent. The same respective b e h a v i o u r s of acylactivated d i h y d r o c o u m a r i n s 3 and 7 have also been observed [5]. Therefore, the rate linfiting step in the i n a c t i v a t i o n of ct-chymotrypsin by these derivatives is likely the acylation step. This last result leads to the following c o m m e n t : t h a n k s to their h y d r o p h o b i c nature, the d i h y d r o c o u m a r i n s insert easily i n the b i n d i n g pocket of ct-chymotrypsin ; their kinetic constants K m are therefore very low. Thus c o u m a r i n is also k n o w n as a good competitive i n h i b i t o r of a - c h y m o t r y p s i n [26]. However their b i n d i n g are largely u n p r o d u c tive as i n d i c a t e d by the lo~v values of koat. This b e h a v i o u r contrasts with the b e h a v i o u r of a n o t h e r sterically restricted substrate, m e t h y l d i h y d r o i s o carbostyril-3-carboxylate [27], the cyclic analogue of the specific substrate, m e t h y l N-acetyl-L-phen y l a l a n i n a t e , w h i c h scissile ester b o n d is exocyclic. The D e n a n t i o m e r of lhis c o m p o u n d (but not the L e n a n t i o m e r ) is a good substrate for a-chym o t r y p s i n (Km = 0.52.7 mM a n d kea t = 22.7 sec -1 at pH 7.9 and 25 ° ) ; its exocyclic ester group is i n d e e d correctly p o s i t i o n e d t o w a r d s the catalytic entities of the enzyme t h a n k s to the correct fit of its t w o - r i n g part in the b i n d i n g pocket of a-chym o t r y p s i n [28]. F i n a l l y all these data are c o m p a t i b l e w i t h the reaction scheme s h o w n in figure 7. After formation of the Micha~lis complex, the enzyme is acetylated on the active serine-195 ; in the t r a n s i e n t acyl-enzyme, the reactive p - h y d r o x y b e n z y l halide is t r a n s f o r m e d into q u i n o n e m e t h i d e [29] w h i c h is i n s t a n t a n e o u s l y attacked either b y a n y nucleophilie e n z y m i c residue located at p r o x i m i t y or by m e d i u m nvater. I n the first case, a chemically modified enzyme is o b t a i n e d ; in the second one, the native enzyme is restored after deacylation of the i n t e r m e d i a t e complex. The n u m b e r of molecules of radioactive dih y d r o c o u m a r i n s [14C] 1 or [3H] 3 i n c o r p o r a t e d in a - c h y m o t r y p s i n at pH 7 a n d 25 ° is slightly h i g h e r t h a n one. The a m i n o acid analyses of the modified enzyme on acid h y d r o l y s e s reveal m a i n l y a loss of 0.75-1 h i s t i d i n e residue. F r o m the data of diagonal peptide maps t h i s h i s t i d i n e has been identified as histidine-57 (at least i n tt-chymot r y p s i n i n a c t i v a t e d by 3), w h i c h is at the active site a n d whose a l k y l a t i o n is k n o w n to change tile
J.-J. Bdchet, A. D u p a i x a n d I. B l a g o e v a .
238
c a t a l y t i c p r o p e r t i e s of t h e e n z y m e . H o w e v e r o t h e r a m i n o a c i d r e s i d u e s i n ( o r n e a r to) t h e a c t i v e s i t e m i g h t b e p a r t i a l l y m o d i f i e d to a h a r d l y d e t e c t a b l e extent, as m e t h i o n i n e - 1 9 2 or tryptopban-215 w h i c h a r e s e n s i b l e to a l k y l a t i n g r e a g e n t s [30, 31].
r a t h e r r e s t r i c t i v e , at l e a s t f o r s i m p l e a l k y l a l i n g r e a g e n t s [:21], a p a r t i c u l a r r e a c t i v i t y a n d a c c e s s i b i l i t y of t h i s a m i n o a c i d r e s i d u e i n t h e i n t e r i n e diate acyl-enzyme may be responsible for this pref e r e n t i a l a t t a c k (**). TABLE II.
E+S~
Inactivation of ,~-chgmotrgpsin by various reagents at p H 7 and 25 ° .
ES~
s~, 195 acyl-enzyme
V%o. I
i o
/ N. CH 2
\ HOCH2
H o ~o
'2,,,~,~ E + P
modified enzyme
FIG. 7. - - P o s t u l a t e d m e c h a n i s m for the r e a c t i o n of a h a l o m e t h y l a t e d derivative of d i h y d r o e o u m a r i n like 1, ~with a - e h y m o t r y p s i n (see text for details). The symbol Null designates some nucleophilic enzymic residue.
Various bifunctional reagents carrying an acylating function and a second reactive chemical group have been previously used against ~-chymotrypsin. The acylating function was generally an activated ester like p-nitrophenyl ester. The second chemical group can be a normal alkylating f u n c t i o n ( l i k e - N H C O C H 2 B r ) a n d , i n t h i s case, p r e f e r e n t i a l a l k y l a t i o n of m e t h i o n i n e - 1 9 2 o c c u r s a f t e r i n i t i a l f o r m a t i o n of a n a c y l - e n z y m e [3'2, 331. T h i s s e c o n d r e a c t i v e g r o u p c a n also b e p h o t o g e n e r a t e d in situ [M, 35], a n d i n s e r t e d i n t o v a r i o u s a m i n o a c i d r e s i d u e s of t h e e n z y m e . At last, as i n o u r c a s e , i t c a n b e g e n e r a t e d a f t e r s c i s s i o n of t h e e s t e r b o n d of t h e r e a g e n t a n d f o r m a t i o n of t h e acyl-enzyme. Thus an activated benzyl halide p r e s e n t i n t h e l e a v i n g g r o u p of a q u a s i s u b s t r a t e w a s s h o ' w n to r e a c t w i t h o n e ( o r s e v e r a l ) t r y p t o p h a n r e s i d u e (s) p r o x i m a l to t h e a c t i v e s i t e of c h y m o t r y p s i n o r p a p a i n [3~, 37] (*). In this work, the activated alkylating function g e n e r a t e d i n t h e a c t i v e s i t e of a - c h y m o t r y p s i n , preferentially modifies hislidine-57. Although the r e q u i r e m e n t s f o r h i s t i d i n e a l k y l a t i o n s e e m to b e (*) Recently, a b i f u n e t i o n a l i n h i b i t o r of c~-ehymot r y p s i n able to l i b e r a t e a e a r b o n i u m ion in the active s i t e of the enzyme h a s b e e n d e s c r i b e d [,38] ; its m e c h a n i s m of action sho~ees some f e a t u r e s c o m p a r a b l e to those reported for h a l o m e t h y l a t e d derivatives of dihydrocoumarins.
BIOCHIMIE, 1977, 59, n ° 3.
Inhibitor
Diisopropyiphosphorofluoridate (DFP) (a) Phcnylmethanesulfonyl fluoride (PMSF) (a) N, N-Diphenylcarbamoyl chloride (DPCC)(b) L-(-tosylamido-2-phenyl) ethyl chloromethylketone (TPCK) (c) Benzyloxycarhonyl-L-phenylalanine bromomethylketone ( Z P B K ) (,) Z-Ala-Gly-Phe-CH~ Cl(d) Dihydrocoumarin 1 (e) Dihydrocoumarin 2 (f) Dihydrocoumarin 3 (g)
k2nd
(M-I sec-l) 45 250 480 7.7 790 100 170 >~ 20000 3O0O
(a) F r o m F a h r n e y a n d Gold [42]. (b) At pH 7,8; f r o m E r l a n g e r et al. [43]. (e) F r o m Sha~v a n d Ruseica [441. (d) At 40 ° ; f r o m M o r i h a r a a n d Oka [45]. (e) This work ; the a p p a r e n t second-order rate c o n s t a n t is det e r m i n e d i n the conditions 'where the reagent concent r a t i o n (97.5 ~M) is very m u c h larger t h a n the e n z y m e c o n c e n t r a t i o n (0.92 10~M). The r e s i d u a l activity of t h e enzyme is tested a f t e r 0.5,-2 m i n u t e s of i n c u b a t i o n time. (f) Too f a s t to be m e a s u r e d ; u - c h y m o t r y p s i n (0.5 I~M) is completely i n a c t i v a t e d a f t e r i n c u b a t i o n for one m i n u t e i n presence of 2.3 ,uM d i h y d r o e o u m a r i n 2. In a n o t h e r ikind of experiment, a - c h y m o t r y p s i n (0.009 ;~M) added at pH 7.5 and 25 ° to a med i u m c o n t a i n i n g 10 mM AcTyrOEt i n 0.5 M NaCI a n d the reagent 2 at a c o n c e n t r a t i o n as law as 0.028 aM is still completely i n h i b i t e d u n d e r one m i n u t e . (g) Approx i m a t e value at pH 6.5 ; f r o m B6chet et al. [5].
I n c o n c l u s i o n , t h e u s e of h a l o m e t h y l a t e d d e r i v a t i v e s of d i h y d r o c o u m a r i n s as i n a c t i v a t o r s of tt-chymotrypsin may be limited by the relative s o l u b i l i t y of t h e s e c o m p o u n d s , t h e i r r e l a t i v e stab i l i t y i n a q u e o u s s o l u t i o n (at p H 7 a n d 25 ° , t h e h a l f - l i v e s of i n h i b i t o r s 1 a n d 3 a r e a b o u t 65 a n d 15 m i n u t e s , r e s p e c t i v e l y ) , a n d t h e n e c e s s i t y to u s e them in a sufficient excess (except for the most s p e c i f i c d e r i v a t i v e 2). H o w e v e r t h e i r e f f i c i e n c y is h i g h . I n t a b l e II, t h e apparent s e c o n d - o r d e r r a t e c o n s t a n t s ( k o b ~ J [ I n h i b i t o r 3 ) f o r i n a c t i v a t i o n of a - c h y m o t r y p s i n b y 1, 2 a n d 3 a r e c o m p a r e d v c i t h t h e s e c o n d - o r d e r r a t e c o n s t a n t s f o r i n a c t i v a t i o n of t h i s e n z y m e b y a fe~v a c t i v e - s i t e - d i r e c t e d r e a g e n t s . It a p p e a r s t h a t h a l o m e t h y l a t e d d e r i v a t i v e s of dihydroeoumarins, e s p e c i a l l y c o m p o u n d s 2 a n d 3, (**) I n model reactions, it h a s b e e n s h o w n t h a t the d i h v d r o c o u m a r i n s 1 and 3 svere able to react w i t h imidazole in aqueous solution, yielding d e r i v a t i v e s s u b s t i t u t e d on the c a r b o n 6 [5, 39].
Inactivation
of a-chgmotrgpsin
are among the most efficient known synthetic i r r e v e r s i b l e i n h i b i t o r s of a - c h y m o t r y p s i n .
by
(< s u i c i d e
This w o r k ~vas supported by grants f r o m CNRS (Groupe de Recherches No. 13) and D6I~gation G6n6tale h la Recherche Scientifique et Technique (Conventions No. 71.7.2627 and 73.7.1687). We are very much indebted to Dr. M. W a k s e l m a n and Professor M. Vilkas for the discussions 'which led to the synthesis and enzymatic application of the h a l o m e t h y l a t e d derivatives of d i h y d r o c o u m a r i n s . We 'wish to t h a n k Dr. J. Van H e i j e n h o o r t and Mrs. A. Mantot for p e r f o r m i n g a m i n o acid analyses. We are indebted to Dr. B. Ribadeau Dumas for p e r f o r m i n g m e t h i o n i n e sulfone analyses. We also w i s h to acknowledge Dr. J. F. Petit 'who made available the use of a high-voltage electrophoresis apparatus. W e are grateful to Dr. C. Ghelis for his advices and useful discussions. We t h a n k Dr. Thusius for carefully reading the manuscript. R~su ~i~. L'c~-chymotrypsine est r a p i d e m e n t et irr~versiblem e n t inactiv~e par une s~rie de d~riv~s halom~thyl~s de d i h y d r o c o u m a r i n e s h pH 7 et 25 °. L'inactivation est d~pendante du pH, et optimale h pH neutre ; elle est aussi plus ou moins totale suivant les concentrations respectives d ' i n h i b i t e u r et d'enzyme. Ces d i h y d r o c o u m a r i n e s sont des s u b s t r a t s pour l'ct-chymotrypsine et, d u r a n t l'acte eatalytique, une f o n c t i o n alkylante latente des r~actifs est activ~e au centre actif de l'enzyme, et r~agit avec tout r6sidu nucl~ophile d'acide anmiu~ voisin. La st,0echiom6trie de la r~action des d~rives r a d i o a c t i f s c o r r e s p o n d a n t s avee 1'enzyme est l~g6rement sup~rieure h un, m a t s une h i s t i d i n e de l'ct-ehymotrypsine est surtout modifi6e. Cette histidine est identifi~e comme l'histidine-57 par la m~thode des cartes peptidiques. P a r r a p p o r t h celle d'autres r6actifs, l'efficacit~ de ces ¢ substrats-suicide >> el, en particulier, celle du d6riv6 2 p o r t e u r d'une chatne lat~rale caract~ristique des substrats de l ' a - c h y m o t r y p s i n e , est tr~s grande. REFERENCES. 1. Sha'w, E. (1970) E n z y m e s , 3rd Ed., 1, 91-146. 2. Powers, J. C..& Tuhy, P. M. (1973) Biochemistry, 12, 4767-4774. 3. Rando, R. R. (1974) Science, 185, 320-324. 4. Sigman, D. S. ,~ Mooser, G. (1975) Ann. Rev. Biochem., 44, 889-931. 5. Bdchet, J. J., Dupaix, A., Yon, J., ~ a k s e l m a n , M., Robert, J. C. & Vilkas, M. (1973) Eur. J. Biochem., 35, 527-539. 6. ~,Va:kselman, M., Hamon, J. F. ,a Vilkas, M. (1974) Tetrahedron, 30, 4069-4079. 7. Nicolle, J. P., Hamon, J. F. & W~kselman, M. (1977) Bull. Soe. Chim. Fr., in the press. 8. B~chet, J. J., Dupaix, A., Roucous, C. ~ Bonamy, A. M. (1977) Biochimie, 59, 241-246. 9. Dixon, G. H. & Neurath, H. (1957) J. Biol. Chem., 225, 1049-1059.
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A c.kn olwledymen is.
substrates
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