Acylation of a-and s-Chymotrypsins by p-Nitrophenyl Acetate ENZYME-SUBSTRATE COMPLEX FOR.MATION AND pH DEPENDENCE*

SUMMARY The acylation step of the cr-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate has been studied at pH 7.1 and 25’ in Z-propanol-water mixtures. By the use of higher substrate concentrations than those used previously under the same conditions, it was shown that, contrary to a previous report, the reaction of acylation does follow saturation kinetics. The binding constant, K,, and the acylation rate constant, Kz, were determined. The acylation step of the &chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate in acetonitrile-water was also shown to follow saturation kinetics. The pH dependences of kz and K, are similar to those reported for or-chymo-trypsin, although the decrease in kz at high pH is less for 6-chymotrypsin. Thus, the pH dependence of acylation by p-nitrophenyl acetate is qualitatively different from that of specific substrates where kz is pH independent and KS increases with pH. An explanation for this peculiar pH dependence of chymotrypsin-catalyzed

From the Division of Biochemistry, Department of Chemistry, Northwestern University, Evanston, SUMMARY The acylation step of the cr-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate has been studied at pH 7.1 and 25' in Z-propanol-water mixtures. By the use of higher substrate concentrations than those used previously under the same conditions, it was shown that, contrary to a previous report, the reaction of acylation does follow saturation kinetics.
The binding constant, K,, and the acylation rate constant, Kz, were determined.
The acylation step of the &chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate in acetonitrile-water was also shown to follow saturation kinetics.
The pH dependences of kz and K, are similar to those reported for or-chymotrypsin, although the decrease in kz at high pH is less for 6chymotrypsin.
Thus, the pH dependence of acylation by p-nitrophenyl acetate is qualitatively different from that of specific substrates where kz is pH independent and KS increases with pH. An explanation for this peculiar pH dependence of chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate is offered.
In the hydrolysis of p-nitrophenyl acetate (8) catalyzed by ol-chymotrypsin (E), a rapid initial liberation of p-nitrophenol, a "burst," was observed (l), followed by a slower, zero order release. The "burst" was shown to be due to the formation of an acyl-enzyme intermediate (ES'). From a kinetic study of the reaction, Gutfreund and Sturtevant (2) concluded that the scheme of Equation followed by acylation of the enzyme and then deacylation of the acyl-enzyme intermediate. This mechanism is considered to apply also to the hydrolysis of specific substrates.
The validity of Gutfreund and Sturtevant's kinetic interpretation has been confirmed in this laboratory (3), at lower concentrations of organic solvent and different pH values. However, the original data have later been shown to be somewhat ambiguous due to the restricted range of concentration of the substrate (4).
The catalytic properties of cr-chymotrypsin are severely diminished in the alkaline pH region (5, 6); the second order rate constant, Icot&,,, for the a-chymotrypsin-catalyzed hydrolysis shows a sharp decrease above pH 8.5, with an apparent dependence on an acid group of pK, about 8.8 (7). For specific amide substrates this decrease is due to a change in the binding ability of the enzyme, appearing as an increase in the dissociation con- stant of the enzyme-substrate complex, K, (8-11). When pnitrophenyl acetate is the substrate for cr-chymotrypsin-catalyzed hydrolysis, the binding constant does not change with pH and the acylation rate constant, IQ, decreases at high pH producing also a decrease in the observed second order rate constant (7, 10).
The validity of this difference in behavior between p-nitrophenyl acetate and specific amides has been questioned (9) since at higher buffer and organic solvent concentrations than those previously used (3,7), Faller and Sturtevant (4) failed to confirm that the acylation step of the cu-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate follows Michaelis-Menten kinetics (Equation 1). The latter authors pointed out that they were unable to separate the observed rate constants into kz and K, by the usual Eadie plots (14) possibly because the highest sub-strate concentration was considerably less than K,. At the beginning of this study we hoped that by using higher concentrations of the substrate we could obtain values of kz and K, and thereby confirm that an enzyme-substrate complex is formed in the a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate, even under the conditions used by these authors.
By studying the pH dependence of binding constants of specific substrates (12), and inhibitors (13), we have established the existence of a significant difference between the alkaline pH dependences of CY-and &chymotrypsin-catalyzed hydrolyses: the second order rate constants and substrate binding ability decrease much less at high pH for &chymotrypsin than for Qchymotrypsin.
Therefore, in the present study we have also investigated the acylation of &chymotrypsin by p-nitrophenyl acetate in order to determine whether the S-chymotrypsin-cata- lyzed reaction also would obey Equation 1 and to compare the distilled water and analytical grade reagents. For the experi-pH dependence of this reaction with that for a-chymotrypsin. ments with ar-chymotrypsm the Tris buffers contained 0.1 M NaCl.

EXPERIMENTAL PROCEDURE
Methods--The pH of each buffer solution was measured before Materials-or-Chymotrypsin (lot CDI9KH) and 6-chymo-and after reaction using a Corning 12 research meter or a Radiomtrypsin (lot CDD-6032) were three times crystallized, salt-free eter 4c with type B glass electrode. Buffer solutions for preproducts from Worthington. Spectrophotometric titrations with steady state measurements were made up twice the required N-trans-cinnamoylimidazole (15) at 335 nm indicated that the final concentration and the pH quoted refers to that after mixing. enzymes had activities corresponding to 80% and 83% purity, The solubilities of p-nitrophenyl acetate in 2-propanol-water respectively. p-Nitrophenyl acetate (Aldrich) was recrystal-and 2-propanol-buffer mixtures were determined from the conheed from absolute ethanol and chloroform-hexane, m.p. 80". centration of saturated solutions in equilibrium with the solid Stock solutions of the substrate were freshly prepared in 2-phase. Ester concentrations were measured spectrophotometpropanol (Baker, analyzed spectrophotometric grade) and rically after alkaline hydrolysis. acetonitrile (Mallinckrodt nanograde). Buffer solutions were The rates of the pre-steady state hydrolysis of p-nitrophenyl prepared according to standard procedures (16)  spectrophotometrically on a Durrum-Gibson stopped flow spectrophotometer at 400 nm, 25.0". Equal volumes of enzyme in buffer and substrate in 2-propanol-water or acetonitrile-water (double final organic solvent concentration) were mixed in the instrument producing a solution of the required concentrations and pH. Mixing effects were shown to be absent by carrying out blanks in the absence of substrate and enzyme. "Burst" rate constants were determined by extrapolating the steady state portion of each reaction to zero time and plotting the logarithm of the difference between the measured absorbance and extrapolated absorbance against time (17). Deacylation rate constants were determined from measurements on a Cary 14 PM recording spectrophotometer at 400 nm, 25.0°, using concentrations of substrate and enzyme similar to those produced after mixing in the rapid reactions. For both methods, rate constants were calculated by an unweighted least squares analysis using a CDC 6400 computer.

AND DISCUSSION
Solubility of p-Nitrophenyl Acetate-The solubility of p-nitrophenyl acetate in 2-propanol-water and 2-propanol-buffer mixtures was measured in order to determine the maximum substrate concentration that could be used for kinetic experiments. The results are shown in Table I. The controlling factor was found to be the solubility in the 2-propanol-water solution before mixing. The highest concentrations used are therefore less than half these solubilities.
Kinetics of Deaylattin of Acetyl Chymotrypsins-Deacylation rate constants, ka, were determined from the steady state rate constants (JG,& at various substrate concentrations. The The values of k) determined at two solvent concentrations are given in Table II. The error in kl is estimated to be less than 10% and this is insignificant in the subtraction of k) from the burst rate constant which is 7 to 200 times greater.
Kinetics of Acylution of a-Chymotrypsin The values of k2 and K, obtained from these plots have standard deviations of less than 10%; they can therefore be taken to be reliable indicators of the saturation of the enzyme. The data previously obtained in this system by Faller and Sturtevant (4) are plotted in Figs. 1A and 2A after correction' to allow for the small difference in pH so that they can be compared with the present work. These points clearly agree with our data and fit well with the calculated curves. These data are therefore also consistent with saturation kinetics, although their limited range of substrate concentrations would not prove the validity of Equation 1 in this system.
The present work was carried out at high buffer concentration at which the deacylation rate constant, ka, and the binding constant, K,, are higher than under the conditions used in previous studies (3, 7, 10). At lower buffer concentration, the substrate concentration could be made greater than K, and, in addition, kg, which must be subtracted from the observed burst rate con-'This small correction (<lo%) was calculated using an unpublished procedure (L. J. Brubacher and F. J. Kezdy) based on a generalized plot of log (kz/kz(lim)) versus pH-pK, and apK, of 7.0. Acylation of Chymotyypsins by p-Nitrophenyl Acetate Vol. 246,No. 19  of the acylation and deacylation rate constants, Ice and k~, respectively, were determined as described for c+ chymotrypsin.
Plots of k. -JCO against substrate concentration and lzO -ka against (,r~ -ka)/[S] at pH 7.91 are shown in Fig.  3. From the latter plot and similar plots at other pH values, value; of kz and K, were calculated using a least squares program and are given with their standard deviations in Table III. The curvature of the plot of k. -ka versus substrate concentration and the finite values of kg and Ii, show that saturation kinetics is followed in the acylation of d-chymotrypsin by pnitrophenyl acetate. A kz versus pH profile from the data of Table III is shown in Fig. 4; included for comparison is the kz versus pH curve for the acylation of cr-chymotrypsin by p-nitrophenyl acetate (7). For &chymotrypsin, K, is almost pH independent. kz decreases at high pH, although this decrease is considerably less than that observed for cr-chymotrypsin.
This difference between the two enzymes in the high pH range is in agreement with previous studies using specific substrates and inhibitors (9, 12, 13), namely that the activity of &chymotrypsin decreases less at high pI1 than does that of ol-chymotrypsin.
Possible explanations and significance of the enhanced stability of d-chymotrypsin over oc-chymotrypsin at alkaline pH have been discussed previously (12,13).
Hypothesis Regarding pll Dependence of Aylation of Chymotrypsins by p-Nitrophenyl Acetate-The pH dependence of the binding constant, K,, of p-nitrophenyl acetate to oc-chymotrypsin differs greatly from that of a specific substrate or inhibitor; in addition the pH dependence of the acylation rate constant, kz, differs from that of a specific amide substrate.
These differences will be related to the different size and structure of p-nitrophenyl acetate.
The binding of specific substrates, such as N-acetyl tyrosine methyl ester, is considered to be controlled (20, 21) principally by the interaction of the aromatic side chain with its binding site, the ar site of Cohen et al. (21). The interactions of the other functionalities of the substrate Iv-it11 their binding sites ensure that the L substrate has one predominant form of binding. At high pH, a conformational change (9, 22) removes the interaction of the side chain with the ar site and K, increases greatly; binding of a specific substrate or inhibitor displaces the conformational equilibrium, with proton uptake (13, 22-24), producing the active conformation.
Therefore 7cz remains constant at high pH while K, increases.
p-Nitrophenyl acetate is much smaller than a specific substrate and lacks the &aryl functionality and is therefore likely to have more than one binding mode. It may also be possible for more than one molecule of the substrate to bind to the enzyme at the same time. We suggest that the conformational change in the enzyme which disrupts the aromatic binding site does not affect the binding of p-nitrophenyl acetate; the molecule is able to bind equally well to the active (EH+) and inactive (E*) forms of the enzyme.
Consequently R, does not exhibit a pH dependence at high pH. In addition the binding of p-nitrophenyl acetate would not cause a shift in the conformational equilibrium since the equality of Z&E and KsBfr requires that the pK, values of E and ES be the same. Therefore the observed acylation rate constant at high pH will be t,hat of the inactive form. The pH dependence of kz requires that this be less than that of the low pH form and this is consistent with the suggested movement (25) of the carboxylate group of aspartate-194 which is held in an ion-pair with the protonated amino terminal of the isoleucine-16.
Such a movement would disturb the sensitive arrangement of serine-195 and histidine-57 needed for catalysis (26). Thus, the pH dependence of the acylation of chymotrypsins can be represented by the scheme of Equation 4