Use of IV-Benzoyl-L-tyrosine Thiobenzyl Ester as a Protease Substrate HYDROLYSIS BY a-CHYMOTRYPSIN AND SUBTILISIN BPN’

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5’-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium.

From the Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003 In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis (2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent K, values of 0.02 and 7 mM and k,,, values of 37 SC' and 126 s-l were observed for the hydrolysis of BzTyrSBzl by cy-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the a-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by a-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on a-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.
In studies on the intracellular proteolytic enzymes of Bacillus subtilis 168 (trp-), we previously reported the use of partially purified N-benzoyl-L-tyrosine thiobenzyl ester to select for a protease-deficient mutant (1). Upon devising a method for the synthesis of the pure compound in good yield, herein reported, we undertook to measure the kinetics of hydrolysis of this thiol ester by a-chymotrypsin and by subtilisin BPN'. The kinetic results obtained with cY-chymotrypsin suggested the presence of a secondary hydrophobic pocket, contiguous with the active site region. This idea was further supported by kinetic studies with the inhibitor indole and by observations made with molecular models of BzTyrSBzl' and trypsin, a protease which has a polypeptide backbone structure closely similar to that of cu-chymotrypsin. Some of the kinetic data for cu-chymotrypsin reported in the literature are discussed in terms of this postulated secondary hydrophobic site.  C 70.59,H 5.42,N 3.58,S 8.19 Found: C 70.20,H 5.64,N 3.61,S 7.96 Upon storage at 0" over an 8-month period, 2 to 3% of the ester was hydrolyzed.
The accumulated free thiol could be eliminated by treating an acetone solution of the compound with 1% hydrogen peroxide, quenching with manganese dioxide, and filtering through a small pad of diatomaceous earth. The subsequent acetone solution remained thiol-free over a a-month period at room temperature. Similar procedures were employed in the syntheses of the ethane thiol and p-thiocresol esters of N-benzoyl-L-tyrosine. Preliminary assays indicated that these esters were less readily hydrolyzed than BzTyrSBzl, and thus they have not been studied further. Spectrophotometric Assay-Both enzymatic and spontaneous hydrolysis of the thiol ester could be followed conveniently by allowing the released benzyl mercaptan to react directly with 5,5'-dithiobis (2nitrobenzoic acid) and observing the increase in absorbance at 412 nm as described for the assay of arylesterases (5) (EC 3.4.4.16) were found to hydrolyze N-benzoyl-L-tyrosine thiobenzyl ester at significant rates. Fig. 1 shows the ranges for which the initial rate increased linearly with the concentration of Lu-chymotrypsin and subtilisin BPN'. As little as 2 rig/ml of a-chymotrypsin and 15 rig/ml of subtilisin BPN' can be measured at an initial substrate concentration of 0.32 mM. The former value is closely comparable to the most sensitive assay yet reported for chymotrypsin (lo), that measuring the hydrolysis of N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester described by Martin et al. (11).
The increase in initial velocity as a function of increasing thiol ester concentration was determined for cY-chymotrypsin and subtilisin BPN'. These data are plotted in Fig. 2 according to the equation first described by Hanes (12). From the slopes and intercepts of these plots we have calculated the apparent K, values and catalytic rate constants, &, shown in Table II, values similar to these were observed in replicated runs.
The hydrolysis of BzTyrSBzl by trypsin has not been studied extensively, but enzyme concentrations of 2 pg/ml gave readily detectable rates under the standard assay conditions, at a BzTyrSBzl concentration of 0.3 mM. This degree of nonspecificity is not as large as for some other substrates. Inagami and Sturtevant (16) have reported that trypsin hydrolyzes N-ace-tyl+tyrosine ethyl ester at 13% the rate observed with chymotrypsin, and Martin et al. (17) observed that trypsin hydrolyzed ZTyrONp at about 40% the rate observed with Lu-chymotrypsin. That the rate observed in the present work was not due to a contamination of trypsin with chymotrypsin is strongly suggested by the fact that the K, for the hydrolysis of BzTyrSBzl by trypsin was indeterminately high. In accord with the original aims of this work, it has been found that BzTyrSBzl can be used to follow the activity of the  (12). The reaction mixtures contained in a total volume of 3 ml: 20 pmol of Tris-Cl, pH 8.0, 0.2 pmol of 5,5'-dithiobis (2-nitrobenzoic acid), amounts of BzTyrSBzl as indicated, 30% acetone (v/v), and either 2.6 x 10-r' mol of or-chymotrypsin or 2.2 x 10-l' mol of subtilisin BPN'. Enzyme concentrations were determined spectrophotometrically as indicated in Fig. 1, and other conditions of assay are as described in Fig. 1. FIG. 3 (left). The effect of indole on the cu-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl (BTTBE) plotted according to Lineweaver and Burk (20). The reaction mixtures contained in a total volume of 3 ml: 20 pmol of Tris-Cl, pH 8.0,0.2 pmol of 5,5'-dithiobis(2-nitrobenzoic acid), amounts of BzTyrSBzl as indicated, 30% (v/v) acetone, 0.3 pg of cY-chymotrypsin, and 0 or 15 rmol of indole. The reactions were run in sealed cuvettes; they were initiated, after equilibration to 30", by addition of enzyme. Corrections were made for a very slight indole-catalyzed hydrolysis of BzTyrSBzl. 0, control; A, 5 mM indole. FIG. 4 (right). The effect of indole on the cu-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate (pNPA) plotted according to Lineweaver and Burk. The reaction mixtures contained in a total volume of 2.7 ml: 100 pmol of morpholinopropane sulfonic acid, pH 7.5, 15% (v/v) acetone, amounts of p-nitrophenyl acetate as indicated, 0.5 mg of cY-chymotrypsin, and 0 or 15 pmol of indole. After thermal equilibration at 30", the reaction was initiated by addition of enzyme, and the optical density increase at 400 nm was measured. The extinction coefficient for p-nitrophenol under the conditions used here was determined to be 12,950 M-' cm-'. This value was used to compute the concentration changes reported in this figure. 0, control; A, 5 mM indole.
classical competitive inhibitor toward BzTyrSBzl (Fig. 3), it behaved as a mixed-type inhibitor (21) toward p-nitrophenyl acetate (Fig. 4). At a final concentration of 5 mM, indole increased the apparent K, of chymotrypsin for p-nitrophenyl acetate from 4.2 x 10m5 to 10.8 x lo-" M, but indole also consistently increased the maximal velocity by 33%, from 9.6 to 12.8 nmol/min. This acceleration of the rate is consistent with the previous observation of Foster (22) that indole may cause up to a 1.6-fold increase in the deacylation step in the hydrolysis of p-nitrophenyl acetate by cu-chymotrypsin. The increased velocity was shown not to be due to indole-catalyzed hydrolysis of p-nitrophenyl acetate; this rate was never more than 3% of the enzyme-catalyzed reaction. With BzTyrSBzl as the substrate the maximal velocity was estimated to be 28.6 nmol/min in the presence or absence of the inhibitor.

Observations Made with Molecular
Models-Using a molecular model of BzTyrSBzl, built to the same scale as Labquip models of subtilisin BPN' and trypsin, we have attempted to fit the substrate into the active site region of these enzymes. With the model of subtilisin BPN' and using the x-ray work on model peptides reported by Robertus et al. (23) we were able to fit the BzTyrSBzl substrate in a plausible way into the active site region. The benzyl group did not appear to occupy any obvious subsite on the enzyme.
In making the same types of observations using the trypsin model, as an approximation to cY-chymotrypsin (24), we were immediately struck by the fact that when the tyrosine was positioned in the tosyl hole such that the tyrosine amide nitrogen could hydrogen bond to the carbonyl of Ser-214, the thiobenzyl group fit very neatly into what appeared, in our model, to be a shallow hydrophobic pocket. Assuming that a similar steric arrangement exists in a-chymotrypsin, the pocket in a-chymotrypsin would be lined by . The latter 2 residues appear to comprise the entrance to this hydrophobic cavity. A schematic representation of the subsites of cy-chymotrypsin following the suggestion of Cohen and Lo (25) and including the newly postulated hydrophobic site (hp) is shown in Fig. 5.
Of these dyes, p-nitro blue tetrazolium proved to be most sensitive and was examined further. Table III shows that the minimum detectable concentration of benzyl mercaptan is 5 x lo-' M. We have successfully used nitro blue tetrazolium to detect thiol esterase activity in polyacrylamide disc gels but have not been able to use it in screening for mutants because colonies of B. subtilis on agar plates were found to reduce this dye in the absence of substrate; however, benzyl mercaptan causes a detectable precipitate with a 2% solution (w/v) of AgNO, (pH 7.0) at concentrations down to 20 pM.
Prior to the present work, Frankfater and Kezdy (26) had reported studies on the hydrolysis of p-nitrophenyl thiolacetate by cy-chymotrypsin and Goldenberg et al. (2) and Polgar (27) had reported the synthesis and studies on the hydrolysis of the more specific substrate N-acetyl-m-phenylalanyl thioethyl ester, respectively.
Very recently during the course of the present work, Hirohara et al. (14) described some detailed kinetic studies on the hydrolysis by a-chymotrypsin of two specific substrates, the thioethyl and p-nitrothiophenyl esters of N-acetyl-L-tryptophan.
Each of the studies cited measured the hydrolysis of the thiol esters by following changes in the ultraviolet or the visible absorption of the p-nitrophenyl thiolate ion.
The use of Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) to assay the thiol released during the hydrolysis of BzTyrSBzl by a-chymotrypsin or subtilisin has provided a convenient continuous assay at visible wavelengths which is comparable in sensitivity for a-chymotrypsin to the most sensitive assay yet reported (11). In addition the thiobenzyl ester has the advantage of being very much more stable than the p-nitrophenyl esters, and presumably also the p-nitrothiophenyl esters, toward spontaneous hydrolysis at alkaline pH values (11). If one wished to assay for hydrolysis of the thiol ester at pH values below neutrality, which is not possible with Ellman's reagent, presumably one of the pyridyl disulfides described by Grassetti and Murray (28) could be used as the chromogenic agent. In discussing the data presented in Table II several points may be noted with respect to a-chymotrypsin.
As shown there, Folk and Schirmer (13) found that the k,,, for BzTyrOEt was 43 s-l, which is very similar to what we report for BzTyrSBzl. On the other hand the apparent K, is 150-fold lower for BzTyrSBzl than for BzTyrOEt. A similar relationship was observed by Hirohara et al. (14) when they compared the steady state kinetics of AcTrpOEt with those of AcTrpSNp, although in this case the K, for the thiol ester was only 14-fold lower than that of the oxygen ester. Do these apparent K, FIG. 5. Schematic representation of the active site region of cychymotrypsin after Cohen and Lo (25). From studies with enzyme and substrate models we propose the existence of a secondary hydrophobic site, designated in this figure as hp. The hp site is proposed to be formed by  are suggested to form the neck to this hydrophobic pocket, such that they are in close proximity to the sulfur residue when BzTyrSBzl is bound to cY-chymotrypsin. ar, /3-aryl; am, cu-acylamide; h, a-hydrogen; n, hydrolytic; hp, hydrophobic. Gutfreund and Hammond have found that the value of the "true" K, (that is, k-, + kJkJZ of BzTyrOEt for cu-chymotrypsin is not more than about 3 times larger than the apparent K, at a pH of 7:2 in 20% isopropyl alcohol (29). Thus, rough estimates for the values of K, are 0.3 mM and 8 mM for BzTyrSBzl and BzTyrOEt, respectively. As the amount and nature of the organic solvents used by various workers can make significant differences in the K, values observed, these values must be taken as only approximations. Insofar as the approximations are valid, we infer that there is a relatively important hydrophobic binding site on the carboxyl side of the aromatic amino acid-binding pocket of a-chymotrypsin. Indeed, Hirohara et al. (14) have interpreted the lower K, values of the sulfur esters compared to the oxygen esters in terms of the greater hydrophobicity of the sulfur relative to oxygen.
In consistent with the studies of the molecular models which suggested there was no site on subtilisin BPN' which could specifically accommodate the benzyl group. More than 10 years ago Jones and Niemann (30) suggested that hydrophobicity of the carboalkoxy group of amino acid esters may contribute significantly to the ability of the ester to bind Lu-chymotrypsin.
More recently a number of workers have presented evidence which may be interpreted in terms of a secondary hydrophobic site on cu-chymotrypsin; their observations are briefly summarized. Zerner and Bender (31) found the K, for N-acetylglycine ethyl ester was 96 mM whereas that for the corresponding p-nitrophenyl ester was 2.2 mM. Similarly, Fastrez and Fersht (32) have reported that the apparent K, of a-chymotrypsin for N-acetyl-L-tyrosine amide is 32 mM whereas that for the corresponding p-nitroanilide is 0.21 mM; they interpret these and other binding data as providing evidence that the anilide portion of the substrate binds nonproductively in the tosyl hole. Alternatively, we suggest their data may be interpreted to imply the presence of a second hydrophobic binding site for the p-nitroanilide (see below). Steitz et al. (33) have presented x-ray difference patterns which show that N-formyl-p-iodophenylalanine binds near the active center but not in the substrate (tosyl) binding pocket. East and Trowbridge (34) have provided calorimetric evidence for the binding of 2 mol of N"-p-toluene sulfonyl-L-arginine methyl ester per mol of cY-chymotrypsin, but of only 1 mol of this substrate per mol of the zymogen. Finally, Berliner and Wong (35) have deduced three binding modes for spin-label substituted phenylsulfonyl fluorides, one of which is outside the substrate binding pocket.
Examination of the molecular models of BzTyrSBzl and trypsin made plausible the suggestion of Hirohara et al. (14) that it was the greater hydrophobicity of sulfur which accounted for the increased binding of the sulfur analogue of N-acetyltryptophan ethyl ester. We observed that the LYchymotrypsin residues Ileu-99 and Val-213 would be in close proximity to the sulfur atom when AcTrpSEt is bound to the enzyme. We feel that it is the binding of the thiobenzyl group at the proposed hydrophobic pocket which in part accounts for the relatively low apparent K, observed with BzTyrSBzl.
Initially we were surprised by the large differences in the apparent K, values for compounds like N-acetyl-L-tyrosinep-chloroanilide (0.67 mM) and the corresponding p-methoxyanilide (12 mM) observed by Inagami et al. (36) and also more recently by Fastrez and Fersht (32). From model building studies, however, we are led to suggest that the methoxy compound does not bind as well simply because the methoxyanilide does not fit well sterically in the secondary hydrophobic pocket. The thiobenzyl group of BzTyrSBzl, because of the possibility of rotation about the methylene, is much more readily positioned in the pocket than the more rigid anilides. We observed that all of those anilides with relatively larger substituents (such as methyls or methoxys) were simply difficult to position in the pocket.
In crystallographic studies indole has only been observed to bind in the tosyl hole of cu-chymotrypsin (33,37) even when the inhibitor enzyme complex is prepared in a saturated solution of indole. The fact that indole is a strict competitive inhibitor against BzTyrSBzl, but actually increases the V,,, when p-nitrophenyl acetate is used as the substrate, suggests that there are at least two partially interacting hydrophobic sites on cu-chymotrypsin and that one of them may be the binding site of the thiobenzyl group of BzTyrSBzl.
If, at the high concentra-tions of indole (5 mM) used in these studies, the indole is binding only in the tosyl hole, we suggest that the proposed secondary hydrophobic site may be the normal binding site for the p-nitrophenyl group of p-nitrophenyl acetate. Alternatively the indole may be binding in the secondary hydrophobic pocket, thereby causing the increase observed in If,,,,,. Obviously, other interpretations are not excluded at present. The data strongly imply that indole and p-nitrophenyl acetate can bind simultaneously to one enzyme molecule. These observations on cu-chymotrypsin may be related to the substrate activations which have been noted for some time with trypsin (see for example, Ref. 38).