ARCHIVES OF BIOCHEMISTRY bND BIOYHYSlCS 160, 259-268 (1972)

A Comparison of Deacylation Rates of Para-Substituted Benzoyl-

Trypsins and Chymotrypsins’

CHIH-CHENG WANG AND ELLIOTT SHAW

Biology Department, Brookhaven National Laboratory, Upton, New York 1iQYS

Received December 20, 1971; accepted February 12, 1972

An accelerative effect of ring substituents on the rate of deacylation of benzoyl- chymotrypsin can be correlated with their ability to increase the reactivity of ester bonds to nucleophilic attack (1). In a more extended study including positively charged substituents, it has been observed that a similar correlation generally exists for benzoyl-trypsins with a rho value of 3.24 for deacylation at pH 8.3. This is some- what higher than the value for chymotrypsin, namely 2.68, and indicates a greater sensitivity to inductive effects. Many of the acyl-enzymes have rate constants for deacylation of the same order of magnitude for both enzymes, whether the substitu- ents are uncharged or positively charged, in accord with observations made earlier on uncharged acyl groups (2). However, notable deviations were observed with certain positively charged substituents which were not the same for both enzymes. Thus the p-guanylmercaptomethyl-benzoyl derivative of trypsin (II) deacylated over lO@fold more rapidly than expected from the substituent effect whereas the rate for chymotrypsin was regular. The results with the p-guanidinobenzoyl enzymes (I) was even more striking since deviations were found of opposing nature: chymo- trypsin, accelerated, and trypsin, decelerated.

The importance of such deviations with respect to the possibility of obtaining selective inhibitors of serine proteinases based on stable acyl-enzymes is discussed.

Trypsin and chymotrypsin are closely similar in mechanism (3) catalyzing hydro- lytic reactions by a process involving recognized steps which include the formation of an enzyme substrate complex (ES), t’he transformation of this to an acyl-enzyme (ES’) followed by the hydrolytic breakdown of this int’ermediate to an acid with regen- eration of the enzyme as shown in Eq. [I]. Additional steps may be involved at stages prior to

E+S+ 1

Ef&-+ES’k3\E + pn (1)

“P

acyl-enzyme formation (4). In fact, this ap- pears to be a general mechanism for serine proteinases in which hydrolysis of the acyl-

1 Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Al omit Energy Commission.

enzyme intermediate derived from the active center serine is facilitated by a nearby imid- azole side chain acting as a general base (3). The differences in specificity which char- acterize the individual proteinases result from a selectivity in binding substrate and in acyl-enzyme formation reflecting struc- tural differences among the proteinases in the binding site region of their homologous structures (5).

Acyl-enzyme intermediates may be iso- lated when the rate of acylation greatly ex- ceeds that of deacylation (6), a situation which permits the study of the deacylation step independently of the other steps of the catalytic process.

Prior studies on the effect of structure of the acyl group on the deacylation step have illuminated various aspects of this process. Caplow and Jencks (1) examined the effect of a series of nonionic p- and m-substituents

259

Copyright 0 1972 by Academic Press, Inc.

260 WANG AND SHAW

on t,hc deacylation of benzoyl-chymotryp- sins and found that the rate of deacylation is increased by electron-withdrawing sub- stituents, evidence consistent with a mecha- nism involving nucleophilic cleavage of the ester bond. A few deviations from a linear correlation of substituent effect were attrib- uted to steric hindrance. Other studies of aromatic acyl groups have suggested pertur- bations of ionizing groups in the active center region (7).

Trypsin is inactivated by ethyl p-guani- dinobenzoate (I, R = CZHJ due to the un- usual stability of p-guanidinobenzoyl-trypsin even at pH 8 (8). The corresponding nitro- phenyl ester has, in consequence, proven to be a useful titrant for trypsin and for en- zymes of similar specificity (9, 10). In order to gain more information about the basis for this slow deacylation and for its possible generality, the guanidino group and other positively charged p-substituenta have been examined with respect to effect on the rate of alkaline hydrolysis of nitrophenyl benzoates and on the deacylation of benzoyl-trypsin and chymotrypsin.

MATERIALS AND METHODS2

Bovine or-chymotrypsin, three times crystal- lized, was obtained from Worthington Biochemical Corp. &Trypsin was chromatographically purified (11) from twice crystallized, salt free, lyophilized bovine trypsin also from Worthington.

Deionized distilled water was used. Organic solvents were of reagent grade, dried over molecu- lar sieves prior to use.

Literature methods were employed for the synthesis of the nitrophenyl esters of p-guanidino- benzoate (9), p-N3-methylguanidinobenzoate (12), p-Na, N3-dimethylguanidinobenzoate (12)) p-N8- methylthioureido-benzoate (12), p-(S,N3-di- methylisothioureido-benzoate (12), and p-chloro- benzoate (13). p-Nitrophenyl p-amidinobenzoate (14) was the kind gift of Dr. Yuichi Kanaoka.

p-Nitrophenyl p’-clminobenzoale. To a mixture of p-aminobenzoic acid (1.0 g) and p-nitrophenol (1.5 g) in 12 ml dioxane was added 1.5 g dicyclo- hexyl carbodiimide. After stirring at room tem- perature for 2 hr, the precipitated dicylcohexyl-

a Melting points were determined with a Fisher- Johns apparatus. Yields of synthetic procedures were not routinely measured since emphasis was given to the isolation of analytically pure mate- rial; generally a minor fraction of each prepara- tion wss adequate for the desired measurements.

urea was filtered off and the filtrate evaporated to dryness under reduced pressure. Unreacted start- ing materials were removed by stirring the residue three times with lo-ml portions of ether. The crude product was taken up in acetone and decolorized with charcoal. The yellowish filtrate, on concen- tration to a small volume, deposited yellow prisms, mp 214-216°C.

Anal. Calcd for C13H1004N2: C, 60.46; H, 3.90; N, 10.85. Found: C, 60.27; H, 4.06; N, 10.80.

p-Nitrophenyl p’-bromomethyl benzoate. To a stirred solution of p-carboxybenzylbromide (10.7 g, 0.05 moles) and p-nitrophenol (7.5 g, 0.054 moles) in peroxide free dioxane (100 ml) was added dicyclohexylcarbodiimide (10.3 g, 0.05 mole). The reaction w&s allowed to proceed for 1.5 hr at room temperature. The filtrate was evaporated to dryness under reduced pressure at 40°C. The solid residue, crystallized from methanol and recrystal- lized from dioxane and methanol, yielded 6.2 g (39%), mp 135-138’C.

Anal. Calcd for G4H1004NBr: C, 50.02; H, 2.99; N, 4.17. Found: C, 49.99; H, 2.86; N. 3.89.

p-Nilrophenyl p’-hydroxybenzoate. Dicyclohex- ylcarbodiimide (2.06 g, 0.01 moles) was added to a stirred solution of peroxide free dioxane (25 ml) containing p-hydroxybenzoic acid (1.38 g, 0.01 moles) and p-nitrophenol (2.09 g, 0.015 moles). After 20 hr at room temperature, the precipitate was filtered and the filtrate evaporated to dryness. The residue was dissolved in chloroform and washed with deionized water continuously until no free p-nitrophenol was removed. The chloro- form layer was then dried over sodium sulfate and evaporated to a heavy syrup. The glass which formed on vacuum desiccation over PzO~ resisted attempts at crystallization. However, the infrared spectrum (Nujol) showed an absorption maximum at 1730 cm-1 (C = 0) and its rate of alkaline hydrol- ysis was consistent with the expected substituent effect. Consequently the preparation, shown to be 61% pure by total color production on alkaline hydrolysis, was judged suitable for use in acyl- enzyme preparation.

p-Nitrophenyl anisate. To a mixture of anisic acid (1.52 g, 0.01 mole) and p-nitrophenol (1.39 g, 0.01 moles) in 15 ml dioxane wm added dicyclo- hexylcarbodiimide (2.06 g, 0.01 moles). It was stirred at room temperature overnight. The pre- cipitated dicyclohexylurea was filtered off and the filtrate evaporated to dryness. The residue was crystallized once from alcohol and twice from ethyl acetate; mp 165-166°C.

Anal. Calcd for ClrHllOsN: C, 61.52; H, 4.06; N, 5.13. Found: C, 60.97; H, 3.96; N, 5.03.

p-Nitrophenyl ester of p’-carboxybenzyldimethyl- sulfonium bromide (II). To a stirred solution of p- nitrophenyl p’-bromomethyl benzoate (1.0 g) in

DEACYLATION OF BENZOYL PROTEINASES 261

10 ml dioxane was added 2 ml dimethyl sulfide. A large deposition of crystals occurred within minutes. After 24 hr at room temperature the mixture was diluted with ether and the crystals collected by suction filtration. The product was recrystallized from N,N-dimethylformamide and ether to yield 1:2 g (633a/,), mp 142-144°C.

Anal. Calcd for C16H16004NSBr: C, 48.24; H, 4.05; N, 3.52; S, 8.05. Found: C, 48.11; H, 3.89; N, 3.47; S, 8.35.

p-Nitrophenyl ester of 2-p’-carboxybenzyl iso- thiouronium bromide. To a solution of p-nitro- phenyl p’-bromomethylbenzoate (0.672 g, 2 mmole) in 5 ml dioxane was added a solution of thiourea (0.167 g, 2.2 mmoles) in 2.5 ml ethanol and 15 ml ether. After 40 hr at room temperature the solid was collected and washed with ethanol- ether (1:2) to give 0.42 g crude product. Crystal- lization from N,N-dimethylformamide, ethanol, and ether gave mp 218-219°C.

Anal. Calcd for ClsHlaOaNISBr: C, 43.70; H, 3.42; N, 10.19; S, 7.78. Found: C, 43.48; H, 3.53; N, 9.95; S, 8.31.

p-Nilrophenyl ester of 2-p’-carboxybenzyl-l- melhylisothiouronium bromide. The foregoing procedure was used with N-methylthiourea in- stead of thiourea; the product had mp 175-176°C.

Anal. Calcd for C16H160*NN3SBr: C, 45.07; H, 3.78; N, 9.85. Found: C, 45.18; H, 3.94; N, 9.55.

p-Nitrophenyl ester of 2-p’-carboxybenzyl-l ,% dimethyl isothiouronium bromide. When t,he fore- going procedure was used with N,N-dimethyl- thiourea, the product had mp 2%221°C.

Anal. Calcd for CIJI,sOdNISBr: C, 46.36; H, 4.12; N, 9.54; S, 7.28. Found: C, 46.38; H, 4.07; N, 9.55; S, 7.40.

p-Nitrophenyl p’-(w-dimethylsulfonio-aceta- mido)benzoate bromide (III). To a solution of p- nit,rophenyl p’-aminobenzoate in 10 ml dry acetone was added 1 ml freshly distilled bromoacetyl bromide. The mixture was refluxed for 10 min, cooled, and the solvent removed by evaporation under reduced pressure at 30°C. The residue was washed with ether to remove excess bromoacetyl bromide and taken up in 5 ml N,N-dimethyl- formamide-ether (1:4 v/v). Two milliliters of dimethyl sulfide was then added and the mixture refluxed for 10 min. Crystals formed on storage at 4°C overnight. Recrystallization from N, N- dimethylformamide-ether provided 0.17 g (lo%), mp 160-161°C.

Anal. Calcd for CDH1705N2SBr: C, 46.26; H, 3.88; N, 6.31. Found: C, 46.02; H, 3.97; N, 6.44.

Preparation of acyl enzymes. Acylation of p- trypsin was generally carried out at room tem- perature (22 f O.l’C) at pH 8.3 by treating a solu- tion of the enzyme (5 mg/ml) in 0.1 M Verona1 buffer containing 0.02 M CaClz with a four- to

TABLE I REACTION OF p-SUBSTITUTED-p'-NITROPHENYL

BENZOATES WITH WCHYMOTRYPSIN AND &TRYPSIN"

Com- pound” (No.)

PH

Reaction time (min) Acvlation

ol-chymo- P-twsin trypsin 1%)

-

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

5.G.h 8.3deu 8.3f.Q 8.3d,i 8.3d+ 8.3f.v 8.3Jsg 5.6.30 5.6e.i 8.3f 90 8.3f.g 5.60.; 8.3dno 8.3f.g 8.3f.i 8.3d.j 8.3dli 8.3fpi 8.3d.h 8.3Jj~ 8.3f .I 8.3dv” 8.3d-” 8.3,s" 8.3f'Q 8.3dpi 8.3dei 8.3f *g 8.3f .g 8.3d-i 8.3dzg 8.3J4 4.4e,t 4.4ca"

0.5 20

60 2

20 3

30

180 70

120 100

20 5

5

1

3

3 3

3 3

3 10

5

2 60

30 30

8 7

4

5

85 96 65 83 91 86 83 45 18 74 81 49 65 57 84 65 59 88 44 96 79 73

100 84 80 20 94 36 89 45 12 86 42 50

a Protein concentration, 5 mgjml; see text for the work up of the acylenzyme intermediates.

b See Table II for structures. c 0.1 M acetate buffer. d 0.1 M Verona1 buffer, 0.02 M CaCL. e 0.1 M acetate buffer, 0.02 M CaC12. f 0.1 M Verona1 buffer. g Compound to protein ratio = 4. h Compound to protein ratio = 10. i Compound to protein ratio = 8. j Same as g except compound was added at two

proportions at 10 min apart. h Same as g except compound was added at

three proportions at equal time intervals.

262 WANG AND SHAW

tenfold excess of the p-nitrophenyl benzoates added as a 10-Z M solution in dioxane or N,N- dimethylformamide. The acylation conditions for individual esters are given in Table I. After acyla- tion, the mixture was quickly adjusted to pH 3 with formic acid, gel filtered through a column of Bio-gel P-2 (2 X 60 cm) with 10e3 N HCl as eluent, and lyophilized.

Acylation of a-chymotrypsin was carried out similarly but without calcium chloride. In the case of a few acyl-enzymes which deacylated rapidly at pH 8.3, acylation was carried out at a lower pH in acetate buffers (Table I). In many cases incompletely acylated preparations were obtained, however this did not affect their suit- ability for deacylation studies since the first order rate constant determination depends on measure- ment of the percent change in activity, not the absolute value.

Rates of deacylation (ks). A solution of the acyl- enzyme intermediate in 10v3 N HCl (3 mg/ml) was added to 2 vol 0.1 M Verona1 buffer, pH 8.3 at 22 f O.l”C and the formation of active enzyme was determined spectroscopically (10) at 410 m/r with the use of titrants. For this purpose, aliquots were removed at various time intervals and treated with p-nitrophenyl p’-guanidinobenzoate in the case of trypsin or p-nitrophenyl p’-(o-dimethylsul- fonioacetamido)benzoate bromide (III, R = p-nitrophenyl) in the case of chymotrypsin. For those acyl-enzyme intermediates which deacylate too rapidly to be measured this way, the deacyla- tion rate was determined by addition of the acyl-enzyme intermediates to the buffer containing titrants and the rate of release of p-nitrophenolate ion was measured.

7 “,-“\ 7

/C-N C-O-R H-N 8

A

H2N\ Hz

+ /c-s-c 43 5-O-R

H2N 0

H3C \ H2 i I-'

+/

CJ-C-C- N-L

H3C

I

II

m

The first order rate constants were obtained by a computer performed least-square treatment of

(a - ~,)/a - Q). The correlation lines in the sigma-rho Hammett plots (15) were also obtained by a computer treated least-square process.

Alkaline hydrolysis of p-nitrophenyl benzoates. The nitrophenyl esters (0.125m~) were hydrolyzed at 25°C in 0.1 M Verona1 buffer, pH 8.3 contain- ing 307& N,N-dimethylformamide and at an ionic strength of 1.0. At intervals aliquots were removed from the incubation mixture and the amount of p-nitrophenoxide formed was determined spectro- photometrically at 410 nm. The pseudo first order rate constants and the slope in the sigma-rho plot were obtained by a computer performed least-square process. The rho value for hydrolysis under the conditions used is represented by the line shown in Fig. 1.

Determination of kinetic constants kz and K, . The kinetic constants for the reaction of chymo- trypsin with the nitrophenyl ester of p-(dimethyl- sulfonioacetamido) benzoate were determined according to the following equations

-bt = In ([El0 - [ES’])/([E],)

‘b = (l)/(h) + ~~J/(kzf%)

K m,ar,oj = (K, kr)/(kz + k3)

k at = (kzka)/(h + M,

where b is the first order rate constant of the pre- steady state reaction at a given substrate concen- tration (cf. Bender et al., 16) and the other con-

FIG. 1. Sigma-rho plot of the alkaline hydrolysis of p-substituted p’-nitrophenyl benzoates at 25%

the data according to equation kl(tz - tl) = In in 0.1 M Verona1 buffer, pH 8.3, ionic strength 1.0.

DEACYLATION OF BENZOYL PROTEINAYES 263

FIG. 2. Deacylation of p-(dimethylsulfonio- acetamido)benzoyl-c-chymotrypsin at pH 3.3 in 0.1 M Verona1 buffer at 22 f 0.05%.

FIG. 3. The effect of reagent concentration on the first order rate constant, b, for the acylation of a-chymotrypsin by the nitrophenyl ester of p- (dimethylsulfonioacetamido) benzoate (III) at pH 8.3, 22°C.

&ants have their usual significance. The deacyla- tion rate, ka , w&5 determined on the isolated acyl-enzyme; kt and k, were determined from a plot of 6 as a function of reagent concentration (Fig. 3) under the same conditions (10, 16).

RESULTS AND DISCUSSION

The rate of alkaline hydrolysis of a series of p-substituted p-nitrophenol benzoates at pH 8.3 in 0.1 M Verona1 buffer containing 30 5% N , N-dimethylformamide were deter- mined. The results are given in Table II to- gether with calculated sigma values. The sigma values for p-OH, p-OCHS, p-CH, and p-H substituents were taken from the com- pilation of McDaniel and Brown (17) and combined with the observed rates of hydrol- ysis under the above conditions to obtain a sigma-rho plot for the hydrolysis of the corresponding nitrophcnyl benzoates as shown in Fig. 1. The rates follow a linear correlation with a rho value of 1.92. Caplow

TABLE II

and Jencks (1) obtained a rho value of 2.04 for the alkaline hydrolysis of p-nitrophenyl benzoates at pH 11.26 in 33% acetonitrile at ionic strength 1.0. Kirsch, Clewell and Simon (18) also studied alkaline hydrolysis of p-nitrophenyl benzoates and reported a rho value of 2.006. Thus, these reported values are in good accord with that derived from the plot in Fig. 1. All of the sigma val- ues of the positively charged benzoates were determined by their rates of hydrolysis and the line shown in Fig. 1. It is recognized that these values assume a linear correlation of substituent effect in alkaline hydrolysis for this group of ionically substituted benzoates which may not be uniformly valid and thus may lead to errors in interpretation of sub- stituent effect in the enzyme deacylation studies.

264 WANG AxI1 SHAW

The rates of deacylation of acyl-chymo- trypsins were monitored by measuring the amount of p-nitrophenoxide released in the initial burst when aliquots of the deacylating mixture were treated with the nitrophenyl ester of p-(dimethylsulfonioacetamido)ben- zoate (III) discovered in this study to be a useful new titrant for chymotrypsin.3 The slow deacylation of the derived acyl-chymo- trypsin is indicated in Fig. 2. To determine whether this reagent satisfied the criteria of a good titrant, that is, could be used validly at a single concentration, the kinetic con- stants of its hydrolysis by chymotrypsin were determined as described by Bender et al. (16). From the variation in presteady state rate constant b with substrate concen- tration (Fig. 3), the kinetic constants given in Table III were calculated. It is evident that Ic% >> i& and that Kmcapp) is sufficiently low to permit use of the reagent at concen- trations well above that value. An added advantage is the failure to give a burst with trypsin thus permitting the titration of chymotrypsin in the presence of tyrpsin (Fig. 4).

The sigma-rho plots for the deacylation of acyl-cY-chymotrypsins and acyl-trypsins are shown in Figs. 5 and 6. In the case of the benzoyl-cu-chymotrypsins, a good correlation between the rate of hydrolysis and the elec- tron-attracting ability is observed for most of the p-substituted derivatives, although the line was drawn to best fit the points for p-H, p-OCHI, p-CH3 and p-OH. Even most of the ionic substituents fall on the line, ex- tending the observations of Caplow and Jencks (1) to a new class of substituents. However, p-guanidino (compound 1) and its monomethyl derivative (compound 2) deac- ylate more rapidly than expected on the basis of substituent effect on ester bond re- activity. In the case of the acyl-trypsins, the positive substituents which deviate from the Hammett relationship are more numerous (Fig. 6) and, in general, the deviations are

a No detailed comparison has been made of the value of this method for the titration of chymo- trypsin relative to others (cf. review by F. J. Kezdy and E. T. Kaiser (1970) in Methods in Enzymology (Perlmann, G. E. and Lorand, L., eds.), Vol. 19, p. 3. Academic Press, New York.

TABLE III KINETIC CONSTANTS FOR REACTION OF p-NITRO-

PHENYL p'-(DIMETHYLSULFONIOACETAMIDO)

BENZOATE WITH CHYMOTRYPSIN AT pH 8.3

K,, M kg, set-’ kr, set? k&a

2.6 X 1O-b 0.56 7.79 x 10-S 7.2 X IO3

3.62 X lo-” 7.79 x 10-S 2.15 X 106

-j IOOSec j--

C

+-TIMEISECONDSI

FIG. 4. The reaction of the nitrophenyl ester of p-(dimethylsulfonioacetamido) benzoate with a- chymotrypsin in the presence (a) and absence (b, c) of 1.5 X lO+ M trypsin in 0.1 M Veronal, pH 8.3 at 22°C. Extrapolation of the absorbance to time zero corresponds to cu-chymotrypsin con- centration of (a) 1.57 X 10S5 211, (b) 1.54 X 10-S M, and (c) 3.13 X lO+ M. Concentration of reagent, 6.6 X 10-S M. The amount of cw-chymot’rypsin used (a) 0.5 ml, (b) 0.5 ml, (c) 1.0 ml.

also in the direction of more rapid deacyla- tion. Examples of this behavior include the isothiouronium compounds, numbers 4-6, as well as the sulfonium derivative, number 10. Conceivably, since tryptic specificity favors the hydrolysis of positively charged substrates, the accelerated deacylation of these positively substituted benzoyl-trypsins might reflect a charge interaction which

-0.6 -0.4 -0.2 0 +0.2 +0.4 +0.6 substrate dimensions, deacylates more slow- m ly than expected also suggests that com-

FIG. 5. Sigma-rho plot of the deacylation of p- pound 10 is not a.ccelerated by occupation of substituted bensoyl-ru-chymotrypsins at 22 f 0.05% in 0.1 M Verona1 buffer, pH 8.3; p = 2.68.

the normal binding site. The ability of tryp- sin to form ternary complexes involving 2 moles of certain substrates and resulting in increased catalytic efficiency (21) suggests the existence of a subsidiary charged site on

.‘I the surface of trypsin which may also be responsible for the activation by amines (22). Possibly interaction with this site is responsi- ble for the more rapid deacylation of the benzoyl trypsins containing substituents 4- 6, and 10.

The unusual stability of p-guanidino- benzoyl-trypsin (S-10) in large part stimu- lated the present investigation. Although both are positively charged, at the pH studied, the guanidino group did not exert an electron-attracting effect through the ring system in contrast to the p-amidino group which had the highest sigma constant of the substituents examined (Table 11). The deacylation rate of p-amidinobenzoyl- chymotrypsin and p-amidinobenzoyl-trypsin (compound 17 in Figs. 5 and 6, respectively)

DEACYLATION OF BENZOYL PBOTEINASES 265

plays a role in determining tryptic specific- ity. However, for reasons summarized by Seydoux and Yon (19) the specificity of tryp- sin is very likely a result of high substrate affinity and accelerated acyl-enzyme forma- tion, rather than selectivity in the deacyla- tion step. Furthermore, the positively substituted benzoyl groups observed to deacylate more rapidly than expected from the Hammett relationship have a somewhat varied geometry which, in the case of the sulfonium compound, number 10, places the positive charge further away from the sus- ceptible bond than would be true for specific substrates. As discussed elsewhere (20) an extended substrate conformation apparently is favored in trypsin catalysis. In compound ;O, the extended conformation is about 1.3 A longer than that for lysine. The fact that the shorter sulfonium compound, number 7, which can more nearly approximate normal

0.0001 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ obey the Hammett relationship quite satis-

-0.6 -0.4 -0.2 0 +0.2 +0.4 +0.6 factorily and represents one of the positively

FIG. 6. Sigma-rho plot of the deacylation of charged benzoyl groups for which the deac-

p-substituted benzoyl-fl-trypsins at 22 f 0.05”C ylation rates for both enzymes were within in 0.1 M Verona1 buffer at pH 8.3; p = 3.24. the same order of magnitude. In view of this

266 WANG AND SHAW

result, the findings with the p-guanidino- benzoyl-enzymes are all the more striking. In the case of trypsin a marked negative deviation in deacylation rate was found (Fig. 6, compound l), whereas with chymotrypsin, a positive deviation was encountered (Fig. 5, compound 1). The deviations are diminished by methylation, the dimethyl derivative conforming to the Hammett re- lationship (Figs. 5 and 6, compound 3). Thus some specific interaction of the guanidino group in the p-guanidinobenzoyl-enzymes must be taking place with the enzyme sur- face resulting in opposing effects on t.he deac- ylation mechanism. A change in the bulk of the group without alteration of charge normalizes deacylation behavior. [As men- tioned above, the ‘Labnormally” rapid deac- ylation of the isothiouronium derivative was also normalized by methylation (Fig. 6, compounds 4-6) .]

The data in Figs. 5 and 6 suggest that the deacylation of acyltrypsins (p = 3.24) is slightly more sensitive to electronic effect than is the deacylation of acyl-cy-chymo- trypsins (p = 2.68). A rho value of 2.1 had been reported for benzoyl-chymotrypsins at pH 7.07 (1).

The rates of deacylation for the nonionic

trypsin and chymotrypsin acyl-enzymes in- cluded in this study are all within the same order of magnit,udc, often differing by less tha,n 50 % (Table IV), an observation which conforms to t’he findings of Bender, Kill- heffer, and Kezdy (2) with a series of non- ionic acyl groups varying in deacylation rate over a range of 105. A majority of the benzoyl enzymes with positively charged para- substituents examined in this study (Table IV) also show a similarity in deacylation rate for the two enzymes, including mono- methyl- and N , N’-dimethyl-guanidino, di- methylsulfonio - methyl, N ,S - dimethyl- isothioureido, and amidino.

Negative deviations from the linear cor- relation have been observed earlier in the deacylation of m- and p- nitrobenzoyl chy- motrypsin and attributed to steric hindrance (1). Another possible explanation may be found in the interpretation of the stability of indoleacryloyl-chymotrypsin, and inter- mediate derived from a poor substrate (23). A structural study of this acyl-enzyme led to the conclusion that the carbonyl group of the indoleacryloyl residue was poorly oriented in t,he acyl-enzyme with respect to attack by an activat’ed water molecule in the normal hydrolytic step. This explanation

TABLE IV COMPARISON OF THE RATES OF DEACYLATION OF SUBSTITUTED BENZOYL-&TRYPSINS AND

SUHSTITUTED BENZOYL-LY-CHYMOTRYPSINS

Substituent ka (minP) ka@-trypsin)

P-trypsin a-chymotrypsin ka(a-chymotrypsin)

p-N-C(=NH)NHz 0.00137 0.35 0.0039 p-N-C(=NH)NHCH, 0.0236 0.039 0.60 p-N-C(=NCHz)NHCHs 0.0172 0.0156 1.1 p-CHzS-C(=NH)NHz 3.49 0.0202 173 p-CHzS-C(=NH)NHCHz 1 .Ol 0.018 56 p-CHsS-C(=NCHa)NHCH, 0.0604 0.0137 4.4 p-CHkWHs)z 0.0233 0.0374 0.62 p-NH-C(=NCH,)SCH, 0.00152 0.0037 0.41 p-NH-C(=S)NHCH, 0.013 0.0089 1.5 p-NH-C(=O)CHzS(CH,)z 0.0502 0.00467 11 p-N& 0.000426 0.00158 0.27 p-OH 0.0012 0.0015 0.80 p-OGHa 0.00335 0.00217 1.5 p-CHS 0.00663 0.00473 1.4 P-H 0.0181 0.0138 1.3 p-Cl 0.125 0.020 6.2 p-C(=NH)NHz 2.31 1.82 1.3

DEACYLATION OF BENZOYL PROTEINASES 267

may also apply to many acyl groups whose deacylation rate appears to be abnormally

clotting (32), kinin production (33), comple-

low as in the case of p-guanidinobenzoyl- ment action (34), and fibrinolysis (35) it appears probable that selective inhibitors

trypsin. These might include, among benozyl derivatives, t,he p- and m-nitro substituted

would be valuable tools in physiology and medicine.

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