Conformational states of chymotrypsin at high pH: Temperature effects on catalysis and binding

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Conformational Temperature FREDERICK

Department

476-464

(197%

States of Chymotrypsin at High pH: Effects on Catalysis and Binding1

C. WEDLER,

of Chemistry,

170,

Cogswell

LAURA S. URETSKY, JOANN CENCULA Laboratory, Received

Rensselaer February

Polytechnic

GREGORY

Institute,

McCLUNE

Troy,

New

AND

York

12181

26, 1975

Studies on the effects of temperature on the chymotrypsin-catalyzed hydrolysis of specific and nonspecific substrates reveal markedly nonlinear Arrhenius and van’t Hoff plots. The investigations include the 6- and a-forms of the enzyme at pH 7.8-9.6 and O-40%. Rigorous error-analysis of the data by computer indicates that these nonlinear responses are not artifactual. By determining the effects of temperature and pH on the relative rates of the acylation and deacylation processes, little or no evidence is found for the nonlinear plots being caused by a change in rate-limiting step as these parameters vary. Model calculations also show that van’t Hoff plots that are concave downward, as frequently observed here, cannot be produced by a mechanism involving a change in rate-limiting step. A variety of extra-kinetic data support the view that conformation changes in the enzyme are the most likely explanation. Detailed comparisons of activation parameters calculated for each enzyme-substrate interaction show clearly that changes in enthalpy values are compensated for by changes in entropy. Relative to the a-enzyme, Gchymotrypsin maintains an active-site conformation more favorable for catalysis over a wider temperature range. Different activation parameters dominate the catalytic process with specific substrates, as compared to nonspecific ones. At high pH the binding process is no longer dominated by apolar interactions even with specific substrates, and the enzyme tends to lose its specificity. Overall, the data indicate that the selectivity and, by inference, the structural integrity of the active site decrease as one proceeds from low to high temperature, from neutral to alkaline pH, from 6- to a-chymotrypsin, or from specific to nonspecific amino acid sidechain groups. These studies also have implications for conclusions drawn from data collected at temperatures other than 37°C.

above 7 are known to vary in different ways. For specific substrates keat is relatively independent of pH changes, while K, increases dramatically. In contrast, for nonspecific substrates, such as those lacking sidechain groups (9, 10, 111, as well as for p-nitrophenyl acetate (12, 131, kcat becomes strongly pH dependent while K, remains much less pH dependent. Detailed investigations of these phenomena have provided compelling evidence for a pH-controlled conformational change, with an effective fla near 8.8-9.0 (7, 14-181, related to the Ile 16-Asp 194 salt bridge. Also, the conversion of 6- to a-chymotrypsin is known to alter the energetics, via the pK,(app), of this multistep alkaline transi-

INTRODUCTION

The substrate-binding process for chymotrypsin is not simple. Dominated by apolar interactions in both the sidechain and acylamino loci (l), it is also pH dependent in both the acid (2, 3) and alkaline (4, 5) regions and may involve multiple rapid steps or conformational equilibria (3, 6, 7, 8). Binding and catalysis at pH values ’ This work was supported in part by grants from the National Science Foundation (No. GU-3162 and GB-34751) and from the Petroleum Research Fund administered by the American Chemical Society (No. 2402-Gl). Portions of this work are from a thesis submitted by L. S. U. in partial fultillment of the requirements of the Master of Science degree in this Department. 476 Copyright All rights

0 1975 by Academic Press, of reproduction in any form

Inc. reserved.

CHYMOTRYPSIN:

CONFORMATIONS

tion (19, 22), due to important changes in protein structure, especially the amino group of Ala 149 (23). In an earlier study (24), we investigated the extent to which specificity requirements are altered by high-pH in CY-and S chymotrypsins. To extend these concepts, in this communication we explore the extent and manner in which such high-pH conformational changes alter the dynamic behavior of the enzyme as a function of temperature. The insights into enzyme mechanism to be gained by such perturbation techniques have been seen with certain substrate types for chymotrypsin (25) and in other enzyme systems, viz. fumarase (26), aspartate transcarbamylase (27), glutamine synthetase (28-30), papain (31, 32), and various other systems (33, 34). EXPERIMENTAL Materials. a-Chymotrypsin and Gchymotrypsin (bovine pancreas, 3x crystallized, salt free) were Worthington products. Stock solutions of these enzymes were prepared in 2x-distilled water. Subsequent concentrations of the enzyme active sites were determined by the method of Schonbaum et al. (35) with N-trans-cinnamoylimidazole on a Coleman 124 spectrometer, immediately after the stock enzyme solutions were made and before and after use in each reaction studied. N-acetyl-tleucine methyl ester was prepared by the reaction of 6.0 g of L-leucine (Cycle) with 3.0 ml of distilled thionyl chloride in 50 ml of distilled methanol. The temperature was kept below 0°C during the SOCl, addition, with rapid stirring. The reaction was then continued for 2 h at 35-40°C with stirring. SOCl,, methanol, HCl etc., were removed in uacuo, 10 ml of methanol was added and removed in VacUo (repeated twice). A pale yellow, oily solid was obtained (36). This crude ester was acetylated in chloroform by adding 7.2 ml of distilled acetyl chloride at 0°C in the presence of 13.0 g of potassium carbonate and stirred overnight at room temperature. Water (100 ml) was then added to give two layers; the chloroform layer was further washed with 50 ml water, dried over magnesium sulfate and evaporated in oacuo to an oil. The oil was sublimed to give crystalline product (37); mp, 43.5-45°C (lit., 43-44.5% (38, 39)). N-acetyl-L-phenylalanine methyl ester (Cycle) was used as received or was prepared as was the leucine substrate. Sublimation of the final product from this preparation yielded a white solid; mp, 8889°C (lit., 88.5-89.5% (40)). N-benzoyl-L-alanine methyl ester was prepared from N-benzoyl-L-alanine (Cycle) and diazomethane

AT

HIGH

477

pH

in ethanol/ether. The CH,N, was prepared with the reagents and apparatus of the Aldrich Dialzald kit. Mp, 58-59°C (lit., 56.5-57.5”C (41)). Methods. Substrate stock solutions were prepared with degassed 0.2 M KC1 in 2x-distilled water with 2 mM phosphate buffer (added for pH-Stat stability) and were used immediately thereafter. In no case did the phosphate alter the observed kinetics. Kinetic studies were carried out on a Radiometer Model l”I”I’IC pH-Stat automatic titrator unit with a Type TTA3 titration assembly. The reaction vessel was constantly flushed with a stream of CO,-free (Ascarite treated) water vapor-saturated nitrogen. The electrodes were Radiometer Type G 202OC and Type K 4112. Standardization of the meter was carried out with standard, temperature-calibrated buffers at pH 6.5, 9.0 and 10.0. Kinetic runs were carried out with 2-ml aliquots of substrate stock solution in a thermostatically controlled (+O.l”) reaction vessel. After pH and temperature equilibration, enzyme was added at the desired concentration and the acid-releasing reaction was followed by pH-Stat addition of standardized KOH (titrated against biphthalate). Each reaction was carried to completion. Data for kinetic runs were analyzed with a ONE RUN computer program kindly supplied by Dr. M. L. Bender and modified for our computer. Copies of this program are available to interested researchers upon request. Its basic functions are similar to those described by Baumann et al. (42). Data supplied to the program included: The individual “absorbance” readings A, (proportional to the amount of added base), the maximum “absorbance”A,, time interval between data points .&, the numbtir of data points n, and “6” value for converting A data to concentrations, as with spectrophotometry. The program then calculated the substrate concentration, S, from [(A, - AJrl, the velocity of the reaction from the slope of a chord drawn between the two data points adjacent to the given AC, liv, llS, v/S, then K,, V, k,,, and statistically derived correlation coefficients for the weighted least-squares tits of the Lineweaver-Burk (l/u vs l/S) and the Eadie-Hofstee (IJ vs u/S) plots. The Eadie-Hofstee plot was printed out by the computer to allow rapid data evaluation and correlation with calculated K, and k,,, values. For each kinetic point reported, at least five separate experiments were carried out. Each value used had a correlation coefficient greater than 0.95/1.0. Activation parameters were then calculated from the Arrhenius plots of Lt. RESULTS

AND

DISCUSSION

The temperature-induced variations in kcat and K, (plotted as K,-’ or R) for (Yand &chymotrypsins are presented in Fig. l-3. Substrates with aromatic (phenylala-

478

WEDLER

ET

AL. -3

30 i

1

I

I

1

A

log k

1

I

I

B

“P’ 20

-

‘A<”

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A\A

logi?

_

A\A 0.

_

(591

\ 0.A

(1.6) ----o-_.

30

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7,

- -0.

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-

(5.1) ;s--

(I.61 ----o-o~o

-0-L

D

-

A/ AIA--GA 20

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C-14 5)

316

328

-

./A' 340 10YOK

352

316

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AHA 6) 340 105/OK

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-

352

FIG. 1. Temperature dependence of k eal anda for the hydrolysis of N-acetyl-L-phenylalanine methyl ester, catalyzed by (Al Gchymotrypsin and (Bl a-chymotrypsin, presented as Arrhenius and van’t Hoff plots with E. and AH values, shown in parentheses, in Calories/mole. Other calculated parameters are presented in Table I. (01, pH 7.8; ( A), pH 9.6. Enzyme was used at 0.2-8.0 x 10eB M, depending on pH and temperature.

nine), alkyl (leucine), and small alkyl (alanine) sidechain groups were studied, as an extension of the pH-specificity studies already carried out with these moieties (24). Using the computer-assisted statistical error analysis to eliminate points with correlation coefficients below 0.95, the data points (five per point shown) agreed so well that the error bars lie within the symbols shown in these figures. Other analyses for error and for data precision and accuracy are discussed below. At pH 7.8 and 9.6, above and below the alkaline transition pKa of 8.8, the parameters related to catalytic activation were calculated and are presented in Table I. Upon general inspection of the patterns in Fig. 1-3, one observes that the kcat data at pH 7.8 show a linear dependence on temperature, except for the case of alanine substrate. At pH 9.6, however, nonlinear Arrhenius plots are the rule rather than the exception for all the substrates. For the aromatic sidechain substrate phenylalanine, R shows less evidence of nonlinearity over the conditions studied than with those substrates with alkyl sidechains, viz., leucine and alanine. With increasing temperature at pH 7.8, E generally in-

creases for the phenylalanine substrate but generally decreases for alanine. This type of response is consistent with an interaction in which apolar forces dominate the binding interactions with specific substrates (43) but depends more strongly on polar interactions with less specific substrates. At pH 9.6, K values are smaller than at pH 7.8 and show a decreasing rather than an increasing trend with increasing temperature. This suggests that above the alkaline transition pH, binding forces are no longer dominated by apolar interactions, even for specific substrates with aromatic sidechains. At pH 9.6 with the phenylalanine substrate (from Fig. 1 and Table I) Gchymotrypsin below its transition temperature (TJ2 appears to behave as does a-chymotrypsin above its T,. Compared to a-chymotrypsin, the &enzyme apparently achieves at a lower temperature a conformation * The term Tt (transition temperature) is defined here for convenience as the temperature at which the two linear portions of the plot intersect and is not intended to imply any specific effect. In fact, the models considered below to explain such changes in slope do not predict sharp “breaks” but exhibit gradual curvatures between the two linear regions (51).

CHYMOTRYPSIN:

CONFORMATIONS

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HIGH

+O/“~O\ 20

-

O4 IO)

(38) -A

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188)

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(0)

log i?

479

chymotrypsin R becomes less sensitive to higher temperatures, whereas, for a-enzyme, E becomes more sensitive and decreases drastically. The breaks in kcat and g plots for the a-enzyme occur at the same T, values, but this does not occur with the a-enzyme. That the breaks or intersection points do not coincide for kcat and K plots does not necessarily imply that completely different processes control each but may reflect a shifted equilibrium upon substrate binding. Such nonlinear behavior is usually considered to indicate functionally important multiple conformational states of an enzyme, with the equilibrium between these forms being dependent upon temperature, pH, and the structure of active-sitebound substrates. Other possibilities are discussed below. The shapes of the Arrhenius patterns show considerable variation: Whereas phenylalanine and alanine catalyses show nonlinear, concave downward plots for kcat at pH 9.6, the catalysis with leucine is monophasic with &chymotropsin and concave upward with a-chymotropsin. This latter type of pattern was in fact the pre-

with a lower AElS but a more negative ASS. Furthermore, it maintains a structure more favorable for substrate binding up to higher temperatures. These combined effects may explain in part the inherently greater activity of Gchymotrypsin with specific substrates at 25°C (14-16). The interactions of alanine substrate with chymotrypsin (Fig. 3) exhibit much more drastic changes in both kcat and R data with temperature under virtually all conditions than one sees with the leucine or phenylalanine substrates (Figs. 1 and 2). The slopes also generally agree with values reported by Kaplan and Laidler (44). The keat and a data for Fig. 3 were collected with substrate levels at 15 mM (pH 7.8) and 30-35 mM (pH 9.61, just below the onset of substrate-activation effects (24). With both enzymes, the kcat and R data show lower Tt values at pH 9.6 than at pH 7.8. Although the kcat data at high and low pH for a-enzyme nearly coincide, the Z? data show a lower Tt at high pH. In this latter regard the (Y- and &enzymes differ significantly. The manner in which K reponds to increasing temperature is also very different: Above the T, for 6

046,)

pH

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(-180) 15-

J

IO-!

I 316

I 328

I

I 340 10’/OK

1

I 352

I 316

328

340 lO”/“)(

352

FIG. 2. Temperature dependence of K cal and R for the hydrolysis of N-acetyl+leucine methyl ester, catalyzed by (A) S-chymotrypsin and (B) a-chymotrypsin, presented as Arrhenius and van’t Hoff plots with E. and AH values, shown in parentheses, in Calories/mole. Other calculated parameters are presented in Table I. Symbols are as in Fig. 1. Enzyme was used at 0.7-4.0 x 10m5 M, depending on pH and temperature.

480

WEDLER

ET

AL.

log k

-2

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25

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log R

(-3 6) oe-‘”

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FIG. 3. Temperature dependence of k cal and K for the hydrolysis of N-benzoyl-L-alanine methyl ester, catalyzed by (A) S-chymotrypsin and (B) o-chymotrypsin, presented as Arrhenius and van’t Hoff plots with E, and AH values, shown in parentheses, in Calories/mole. Other calculated parameters are presented in Table I. Symbols are as in Fig. 1. Enzyme was used at 0.8-4.0 xx 10m4 M, depending on pH and temperature.

dominant one observed for fumarase (26). substrates such as alanine the opposite Calculated parameters. Values of free trend seems to occur. Comparisons of enenergy, enthalpy, and entropy for the cata- tropy terms seem to suggest that, with a lytic process under different conditions are specific substrate, protein ordering occurs presented in Table I. It should be pointed in proceeding from the enzyme-substrate out that K, (or its reciprocal) are not true complex to the transition state. However, binding constants but are equal to K, with a nonspecific substrate, some relative [kk$(h, + kJ]-see Scheme I below. For disordering, perhaps of a partially colthis reason, we have not calculated or com- lapsed active site region, must occur in the pared parameters related to the slopes of catalysis of the chemical reaction. the log K vs l/T plots, except in a qualitaThe binding data @) for leucine show tive way. much more pronounced effects than do k,,, First, at or near 25°C where the vast data-(Fig. 2). The T, values are 25 and 18°C majority of chymotrypsin kinetic and ther- for K with 6- and a-chymotrypsin, respecmodynamic data have been collected, the tively. With the &enzyme the change from activation parameters E, and AGS (Table the low-temperature to the high-temperaI) show the expected increase: Both be- ture species is accompanied by the heats of come larger and more positive due to binding (AH) changing from slightly posichanges in both MS and TASS as one tive (unfavorable) to negative. proceeds from specific to nonspecific subFinally, comparing the calculated pastrates. These results are somewhat in con- rameters above and below the transition trast to the results obtained with a series temperatures in nonlinear Arrhenius of substrates by Bender et al. (45) in which plots, one sees that ASS values tend to changes in the ASS term alone appeared to become smaller or more negative, alexplain the changes in the AGS. though this is not without exception. The Second, with specific substrates, the E, general interpretation is that protein is value is altered mainly by pH and less by less ordered, and thus disfavors catalysis temperature, whereas with less specific at higher temperatures.

(b) pH 9.6

(3) N-Bz-L-Ala-Me-ester (a) pH 7.8

(2) N-Ac-L-Leu-Me-ester (a) pH 7.8 (b) pH 9.6

(1) N-Ac-L-Phe-Me-ester (a) pH 7.8 (b) pH 9.6

Substrate, conditions

-

T

25 40 18 32

25 25

25 4 25

0.31 1.26 0.126 0.40

14.1 14.1

105

10

105

kc&-‘)

ACTIVATION

22.1 10.7 22.1 3.0

12.7 12.7

3.5 11.6 22.2

E,

18.1 18.2 18.3 18.4

15.9 15.9

14.7 16.2 14.7

:;GD-

21.5 10.1 21.5 2.4

12.1 12.1

21.6

11.1

2.9

MS

FOR

(Cal/mol)

AGS

(A) SCT

PARAMETERS

I

-3.4 8.1 -3.3 16.0

3.8 3.8

11.2 -25.9 11.2 -52.5

-12.7 -12.7

-39.5 - 18.5 23.1

Ass entropy units)

-r

Enzyme

12 25 12 25

25 12 25

25 18 25

~GIYMOTRYPSIN-CATALYZED

11.8 5.1 -6.9

-ThS-I

AND

TABLE

0.048 0.28 0.048 0.28

6.0 4.0 6.8

52.5 48.5 91.4

30.0 21.0 30.0 21.0

7.8 3.0 9.7

5.9 16.8 11.7

E,

HYDROLYSES

18.4 18.2 18.4 18.2

16.4 15.9 16.3

15.1 14.8 14.8

(Cal/mol)

AGS

(B) a-CT

29.9 20.4 29.9 20.4

7.2 2.4 9.1

5.3 16.2 11.1

AHS

-11.6 -2.2 -11.6 -2.2

9.2 13.4 7.2

9.8 -1.4 3.7

-TASSS

MS

si “3:

7.3

E

zl

3

F 2 0

22

z

8

g z

5 2 3

40.6 7.3 40.6

-31 -47 -24

-12.3

4.9

-32.9

-( entropy t units)

T

482

WEDLER

Data Analysis and Compensation Effects. The mutual compensation of sizeable entropy and enthalpy changes, resulting in relatively small changes in free energy terms, has been discussed extensively in recent years (7, 46-50). For our data in Table I, plots (not shown) of MS vs ASS were linear, with little scatter, and clearly demonstrate compensation behavior. This correlation held true for data above and below Tt values. Interestingly, the “compensation temperature,” T, (46), was 25°C. No differences in T, were observed for keat data from above and below Tt, when one compares data for different enzyme forms, different substrates, or at different pH values. This is in contrast to observations by Glick (47) in solvent perturbation studies with a-chymotrypsin. As discussed earlier (46, 49, 50), the linearity of the enthalpy vs entropy plots can be artifactual, i. e., it may be simply an error plot, unless the data are very precise. As pointed out by Exner (491, plots of log k 1 vs log kz, at temperatures T, and Tz, better reflect such error as scatter from a straight line. Replots of our data in this manner showed that within or between the linear portions of Figs. l-3, the scatter was such that correlation coefficients of 0.90 for the kcat plots were obtained. Thus, it is quite unlikely that the nonlinearities observed in Fig. l-3 are artifactual. Consequently, the enthalpy-entropy compensation effects are real, although the ultimate basis for this behavior is not clearly resolved. The slopes of the log k, vs log kz were slightly less than 1.0, which has been interpreted previously (49) to indicate a loss of selectivity or specificity at higher temperatures. This agrees with our comparisons above. The plots for Gchymotrypsin showed a slope closer to 1.0 than did those for a-chymotrypsin, indicating that the 6 enzyme maintains greater selectivity at higher temperatures than does the o-enzyme. Models Considered. For reactions catalyzed by chymotrypsin, involving an acylenzyme intermediate, one must consider at least two distinct models which could produce the nonlinear Arrhenius plots above. First, for a multistep pathway such as that shown in Scheme I,

ET AL. E + S&ES*ESk”‘E +p,

+;, [Scheme I]

either kz or k3 may be rate determining for a given set of conditions and substrate. Changes in pH or temperature, however, could cause a change in rate-limiting step, e.g., a change from kz > kB to kZ < kB. If, for example, Arrhenius plots of kz and k3 did intersect at an appropriate temperature, one would observe a change in the Arrhenius slope above and below this intersection point. Since K, (app) = K, 1 kd(k, + kg)] this would also produce a nonlinear plot of l/K, (app) vs l/T. For this “sequential” model, however, the van’t Hoff plot would always be concave upward (51). Alternatively, one may consider conformational equilibria between enzyme species, as shown in Scheme II, with each equilibrium capable of changing with temperature: E+S%ESk”““E+P K*j/

j/K*2

E* + S +E*SeE* xm’

[Scheme III

+ P cat and where E* is an enzyme form differing in its binding and/or kinetic properties from E. As discussed by Han (51), this “conformation” model scheme can give rise to van’t Hoff plots which are concave either upward or downward. Upon comparison of such considerations to the van? Hoff plots in Figs. l-3, as well as spectroscopic evidence from other laboratories (6-8, 521, it seems most likely that the conformational model of Scheme II is the best explanation for the patterns in Figs. l-3. This hypothesis has been further tested by initial velocity experiments in the presence and absence of added nucleophiles (53) with each substrate, carried out at pH 7.8 and 9.6 at above and below the transition temperatures, to separate and determine values of k2 and KS as they contribute to kcat under each set of conditions. In virtually all cases, k, remained greater than KS over a wide range of experimental conditions. Thus, a change in ratelimiting step with temperature is not a likely basis for the observed nonlinearity of the plots.

CHYMOTRYPSIN:

CONFORMATIONS

One other artifactual possibility should be considered as an explanation for the nonlinear plots of Figs. l-3; namely, enzyme dimerization. Data in the literature ((54) and Refs. therein) indicate that at pH values above neutrality and at the enzyme levels used in these studies the monomer:dimer ratio is >lO and that dimer dissociation occurs too rapidly to be kinetically significant, even at extremes of the O-45%! temperature range used here. Finally, one might consider that temperature alters the equilibrium between two substrate-binding states, one more “productive” (catalytically efficient) than the other. The present data do not eliminate this possibility; however, it seems unlikely that the more specific substrates (phenylalanine and leucine) would involve such a mechanism. In a sense this mechanism is perhaps merely a specific case of the more general conformational model (Scheme II). Conclusions By combining variations in pH, temperature, and substrate structure we have probed the nature and requirements of specificity of chymotrypsin in the alkaline pH region. With specific substrates, the most pronounced effects occur in the binding parameters. Alanine, as a case bridging specific and nonspecific behavior (24), shows major transitions in both the kcat and K vs l/T plots. Overall, the conformational model best fits the observed kinetics for the Arrhenius and van’t Hoff plots. These data also are consistent with a variety of other extensive studies on active site-substrate interactions obtained by nmr, epr, and chemical probe techniques (55-58) as well as fluorescence data (7). It is interesting but difficult to compare our data with those values reported by Kim and Lumry (71, since their observations were made spectroscopically in the absence of bound substrate or catalytic events. On a qualitative basis, however, our data agree reasonably well with their schemes and values for coupled ionization and conformational changes at high pH (cf. Fig. 8 and Table V of Ref. (7)). It is worthwhile noting also that this is not the first report of nonlinear Arrhenius

AT

HIGH

483

pH

plots for chymotrypsin kinetics, although these earlier data were interpreted somewhat differently. The kinetic data for D and L-ZV-benzoyl-alanine methyl esters of Kaplan and Laidler (44) can be seen to deviate from linearity. This was discussed in terms of reversible denaturation of the enzyme, which is perhaps another way of looking at Scheme II. Kim and Lumry (7) and also Glick (47) discussed their data in a similar way. Further, the data of Bender et al. for trans-cinnamoyl a-chymotrypsin (451, although not very extensive, when plotted do show nonlinear Arrhenius plots. Finally, a careful study on temperature effects for kS with p-nitrophenyl esters of alkylated furoyl substrates by Klappel-3 has revealed pronounced nonlinear dependences. The present data, taken with data from the above mentioned spectroscopic studies, are consistent with increased flexibility of the enzyme structure, and decreased specificity or selectivity, as one proceeds from pH 7.8 to 9.6, from &chymotrypsin to CYchymotrypsin, or from specific to nonspecific substrates. The data do indicate also that the behavior of this enzyme, much studied at 25”C, may be very different at 37”C, i.e., “physiological” conditions. That is, the specificity may not be as well defined, or other parameters may have been altered. These possibilities obviously provide the impetus for further investigation. REFERENCES M. L., AND KBZDY, F. J. (1965) Annu. Biochem. 34, 49-76. 2. VASLOW, F., AND DOHERTY, D. G. (1953) J. Amer. Chem. Sot. 75, 928-931. 3. SHIAO, D. D. F. (1970) Biochemistry 9, 10831090. 4. JOHNSON, C. H., AND KNOWLES, J. R. (1966) Biochem. J. 101, 56-62. 1. BENDER,

Rev.

5. JOHNSON,

C.

Biochem. 6. HAVSTEEN, 771. 7. KIM, Y.

D.,

Chem. 8. GAREL,

chimie 9. WOLFF,

H.,

AND

KNOWLES,

J. 103, 428-430. B. H. (1967) J. Biol.

Sot. J.-R.,

AND

LUMRY,

R.

(1967)

Chem.

242,769-

(1971)

J. Amer.

93, 1003-1013. AND

LABOUESSE,

53, 9-16. J. P., III, WALLACE,

’ M. Klapper,

R.

J.

personal

B. R. A.,

communication.

(1971) PETERSON,

BioR.

484

10. 11. 12. 13. 14. 15. 16. 17.

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