THE INFLUENCE OF pH ON THE KlNETlC CONSTANTS OF cr-CHYMOTRYPSIN-CATALYZED ESTEROLYSIS*

BY LEON W. CUNNINGHAM AND CHARLES S. BROWN

(From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee)

(Received for publication, November 14, 1955)

Determination and comparisons of the kinetic constants for the reactions between a-chymotrypsin and various simple synthetic substrates have been extensively made. Several improvements in the mathematical interpreta- tion of such reactions have resulted, and valuable insights into the molecu- lar basis of enzyme specificity have been obtained (l-6). The wide useful- ness of oc-chymotrypsin in such studies is attributable to its availability, stability, and apparent purity. Recent investigations (7, 8), however, have raised doubts as to the molecular homogeneity of preparations of cY-chymotrypsin. We have recently reported’ briefly on a kinetic investi- gation into this problem. The present paper presents a more detailed dis- cussion of these studies and includes the results of the extension of these methods to closely related systems. These studies were undertaken in the belief that knowledge of the pH-dependence of the catalytic functions of the enzyme might contribute to the understanding of the relationship be- tween molecular heterogeneity and enzymatic activity. In addition, the quantitative description of pa-dependence in terms of the fundamental kinetic constants of an enzymatic reaction, such as has recently been re- ported by Gutfreund (9) for trypsin, should be of value to an understand- ing of the reaction mechanism.

EXPERIMENTAL

Materials-The a-,2 p-, and r-chymotrypsin preparations used in these investigations were prepared by the ammonium sulfate fractionation pro- cedure of Northrop et al. (10) and were stored in the cold as salt-free, lyophilized powders. Each day, fresh enzyme was dissolved in 0.001 N

HCl and stored at 5-10” for use as a stock enzyme solution. All enzyme concentrations were determined spectrophotometrically at 282 rnp, by us- ing the relation, mg. of N per ml. = 0.0763 X optical density (7).

* This investigation was supported by a research grant from the Division of Re- search Grants, National Institutes of Health, United States Public Health Service.

1 A preliminary report of this work was presented before the Forty-sixth meeting of the American Society of Biological Chemists at San Francisco, April, 1955 (1).

2 Obtained through the generosity of Dr. Jules A. Gladner and Dr. Hans Neurath.

287

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Acetyl-L-tryptophar? and acetyl-L-tryptophan ethyl ester were commer- cial products (Mann Research Laboratories). All preparations of ATrEE that were used in experiments reported here were shown by paper chroma- tography to be free of contamination with ATr. Acetyl-L-tyrosine ethyl ester was prepared according to a published procedure (11). Stock solu- tions of 0.0100 M ATEE and 0.0067 M ATrEE in water could be prepared by warming suspensions of these compounds to 85”. Upon rapid cooling to room temperature, clear supersaturated solutions of these slightly solu- ble compounds were obtained which were stable for periods up to 12 hours. Concentrations of these solutions calculated from maximal enzymatic re- lease of hydrogen ions agreed well with the weight concentrations.

All other materials were of reagent grade. Methods-The usual continuous potentiometric titration method for

following esterolytic reactions was employed (7, 12). The assay mixture consisted usually of 10 ml. of substrate, 0.5 ml. of 2.4 M CaC12, 0.5 ml. of 0.08 M buffer, and 1.0 ml. of enzyme solution. Thus, the final concentra- tion of CaC& was 0.1 M. The dependence of k, and of an apparent K, on the ionic strength of the assay mixture has been noted by several investi- gators (13, 14). The use of CaC& in the concentration mentioned is suffi- cient to prevent erratic results from variations in ionic strength owing to the addition to the reaction mixture of other ionic components such as com- petitive inhibitors, and to give near maximal values for lc,. When the volume of substrate added was varied, the total volume was brought to 12 ml. by the addition of distilled water. The standard base used for titra- tion was usually 0.1 N NaOH, but 0.02 N NaOH was employed in runs in which the substrate concentrations were too low for use of the more con- centrated base.

All pH measurements were made with a Cambridge model R pH meter standardized at pH 4 with 0.05 M potassium acid phthalate.

Results

Determination of Kinetic Constants of ATrEE at pH g-Measurements of the initial velocity of the ar-chymotrypsin-catalyzed hydrolysis of ATrEE were made at several substrate concentrations and at pH 8. The values obtained in one such experiment are plotted in Fig. 1 in the manner of Lineweaver and Burk (15). From the slopes and intercepts, K, may be calculated to be 9.3 X 10V5 f 2.0 moles liter-l. Determination of a K, this small required the use of 0.02 N NaOH as the titration base and repre- sents very nearly the extreme experimental limit of the esterase procedure

3 The following abbreviations are used: ATr, acetyl-L-tryptophan; ATrEE, ace-

tyl-I;-tryptophan ethyl ester; ATEE, acetyl-L-tyrosine ethyl ester; Tris, tris(hy- droxymethyl)aminomethane; ATrNH2, acetyl-L-tryptophanamide; ATNH,, acetyl- L-tyrosinamide.

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L. W. CUNNINGHAM AND C. S. BROWN 289

employed. As will be evident from other data (see below), K, for ATrEE falls almost within the experimental error of the method as it was routinely used for pH-dependence studies.

Determination of pH-Dependence of Ica of ATrEE-Measurements of the entire course of the a-chymotrypsin-catalyzed hydrolysis of ATrEE were made at varying values of pH. Three buffers, Tris-HCl, imidazole-HCl, and cacodylic acid-NaOH were required to cover the desired range. None of these buffers appeared to exert any specific influence on the course the reaction. In these studies of pH-dependence the concentration ATrEE was held constant near 0.0083 M, but three concentrations

of of of

I I 2 4

I ,

I/S x lo-3 1,; x lo-3 2

FIG. 1 FIG. 2

FIG. 1. Effect of initial concentration of ATrEE and of ATr on the initial velocity

of the a-chymotrypsin-catalyzed hydrolysis. l , ATrEE alone; A, ATrEE + 0.0125 M ATr. T 25”; buffer, Tris-HCI; pH 8.0.

FIG. 2. Effect of initial concentration of ATrEE and of ATr on the initial velocity

of the a-chymotrypsin-catalyzed hydrolysis. l , ATrEE alone; A, ATrEE + 0.0125 M ATr. T 25’; buffer, cacodylic acid-NaOH; pH 6.1.

enzyme were required so that reasonable reaction rates might be obtained. The enzyme concentration in runs above pH 7.5 was near 7.45 X 10m4 mg. N ml.?, and the buffer was commonly Tris, while in runs between pH 6.5 and 7.5 the enzyme concentration was near 1.49 X 1OP mg. N ml.-I, and the buffer was imidazole. Below pH 6.5 the enzyme concentration was about 3.75 X 1O-3 mg. N ml.+, and the buffer was cacodylic acid. In all cases the buffer and enzyme concentration regions were overlapped so as to disclose any specific effects attributable to buffer or enzyme con- centration. The data obtained were plotted according to the integrated form of the Michaelis-Menten equation (16)

&et = 2.3K, log z + (a~ - a) (1)

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where a0 = initial substrate concentration, a = substrate concentration at time t, e = enzyme concentration, K, = the Michaelis constant, and k, = the rate constant for the decomposition of the enzyme substrate complex into products and enzyme. The calculations make use of the previously determined value of K,. As already indicated, the contribution of the first order term in Equation 1 for our usual system is quite small, since K, is so low. Nevertheless, its use was necessary, and sufficient, to obtain a straight line plot at longer reaction times at pH values above 7 to 7.5. Thus, there was no indication of product inhibition in this region. Below pH 7, however, a progressive deviation from linearity with decreasing pH was observed, indicating a possible variation in K, for the system. In order to determine whether this was the case, the Lineweaver and Burk procedure was again employed to determine K, at pH 6.1. The results of one such determination are shown in Fig. 2. The value for K, obtained was 8.4 f 2 X 1OP mole liter-l. It is apparent, therefore, that there is no significant change in K, over this pH range and that the kinetic changes detected must be due to some other cause.

The demonstration by Foster and his associates (3-6) of product inhibi- tion by ATr in the system a-chymotrypsin-ATrNHz and the dependence of the degree of inhibition upon pH led us to investigate this possibility. The value for K, for ATr reported by these workers for their system at pH 8 was 10 & 2 X 10-a mole liter-l and, at pH 6.9, 2.0 f 0.3 X 1OP mole liter-l. The pH 8 value is large enough so that the inhibition would normally be below the level of detection in our esterase system. However, by again employing the method of Lineweaver and Burk, K, for ATr was determined in the ATrEE-a-chymotrypsin system at pH 6.1 and at pH 8. The results of these investigations are also given in Figs. 1 and 2. From these graphs K, was determined to be 8.7 f 2 X 1O-3 mole liter-’ at p1-I 8 and 1.7 f 0.5 X 10M3 mole liter-l at pH 6.1. The deviation from linearity in the plot of data at pH 6.1 occurs in the region of the extreme limit of the experimental procedure. This deviation was obtained in every experiment to varying degrees, but the limiting slope of the straight line portion always corresponded to a value of KI near 1.7 X 1O-3 mole liter-l. Since it was apparent that K, for the enzymatic reaction was con- stant, but that variation of the degree of inhibition by reaction products was being obtained, the kinetic plot suggested by Foster and Niemann (6) appeared ideal for more complete characterization of the enzymatic reac- tion. The equation is obtained from the integrated form of the Michaelis- Menten equation for the case of inhibition by one of the reaction products.

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L. W. CUNNINGHAM AND C. S. BROWN 291

By dividing by t, and rearrangement, the equation becomes

a0 - a - = t [ 1 log : he - +- t

1-g (3)

where Kr = dissociation constant for the enzyme-inhibitor compound. Thus a plot of (ao - a)lt versus (log ao/a)/t for a single esterase measure- ment should yield a straight line with a slope equal to (- 2.3K,(l -I- u,JKJ)/- (1 - KS/K,) and with an intercept equal to &e/(1 - K8/KI). When the

2 4 6 8

(log~/t)xlO 2 6 7 8 9

PH FIG. 3 FIG. 4

FIG. 3. The hydrolysis of ATrEE by wchymotrypsin. pH 8.43, 0 ; pH 8.02, 0; pH 7.36, n ; pH 6.38, 0 ; pH 5.72, A; pH 5.41, n (see the text).

FIG. 4. The dependence of the ratio of K, for ATrEE to KI for ATr on the pH of the system. l , a-Chymotrypsin; 0, p-chymotrypsin; A, -y-chymotrypsin; 0, calcu- lated from the values obtained from Figs. 1 and 2. T 25”.

data from the series of experiments at, varying pH values were plotted in this fashion, a family of straight lines of negative slope was obtained. An example of these data is shown in Fig. 3. For high pH values the slope is quite small, as is to be expected from the known values of K, and KI in this region. As the pH decreases below 6.5, however, there occurs a gradual increase in the negative slope of these lines. By utilizing the equations just, described and a value of 9 X 10e5 mole liter-’ for K,, a value of K,/K, was determined for each run. These values are shown in Fig. 4. Though there is considerable scatter in these points, since they represent values near the limit of determination in the system employed, it is clear that there is a progressive decrease in KI as the pH is decreased.

Values of Ic3e may be determined for each experiment from the intercepts of the lines on the a0 - u/t axis. From Fig. 4 it is apparent that 1 - KS/K1 is a negligible correction to the intercept at high pH values and rises only

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292 a-CHYMOTRYPSIN-CATALYZED ESTEROLYSIS

to 6 to 8 per cent at the lowest pH values investigated. Since e is known, lc, may be evaluated for each experiment. Values for lc3 had been previ- ously determined from the initial slopes of the plots of the data according to Equation 1 without other regard to product inhibition. These values agreed, as would be predicted, with those obtained in this manner within experimental error, except perhaps at the lowest pH values. When these values for kS were plotted against pH, it was apparent that jca varied with pH in a manner resembling the titration curve of a weak acid. The maxi- mal value of kB was approached in the pH region between 8.3 and 9. At pH 8.7 the experimentally determined value of 1~3 was found to be 0.77 f

I oo- . .

2 2 80- N-J ;rSO-

L5 0 K 40

ii

20-

/ .

.

FIG. 5

--x-F77 PH

FIG. 6

FIG. 5. The dependence of the degree of esterolytic activity of cy-chymotrypsin upon pH. Substrate, ATrEE; T 25”.

FIG. 6. The dependence of the degree of esterolytic activity of cu-chymotrypsin npon pH. Substrate, ATEE; T 25”.

0.03 mole liter-l min.? mg. N-l ml. From the form of the curve, this value was assumed to be 99 per cent of the maximal k3 of the system. Fig. 5 shows the variation of lc, with pH calculated on the basis of per cent of maximal lc, as described. The line through the experimental points repre- sents the theoret,ical dissociation curve of a weak acid of pIl’ = 6.70. Since the substrate, ATrEE, undergoes no change in structure over the pH region investigated, it seems reasonable to ascribe the behavior ob- served to the enzyme.

Another series of reactions was studied at varying pH levels in which all the conditions were the same as those described above, except that the temperature was maintained at 14” instead of 25”. The resulting decrease in rates required that all enzyme concentrations be approximately doubled. Again, the data were examined as a function of pH and the highest value of Jc3, 0.37 f 0.02 mole liter-’ min.? mg. N-l ml., was set as being 99 per

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L. W. CUNNINGHAM AND C. S. BROWN 293

cent of the maximal theoretical value. In this case the experimental data are best described in terms of the dissociation constant of a weak acid of pK’ = 7.00. Although the scatter is somewhat more than was obtained in the previous case, there is no doubt that the entire curve has been dis- placed to higher pH values, as would be expected if the variation does represent a pH-dependent change in ionic form of the enzyme.

Determination of pa-Dependence of ii3 of ATEE-A series of esterase assays was performed which differed from previous determinations in that ATEE was used as substrate. The reactions were studied at 25” with the use of lower concentrations of enzyme, since k3 for ATEE was known (2) to be higher than for ATrEE. In a similar system, k, for ATEE was reported (7) to be 7 X 10M4 mole liter-‘. Upon reexamination in the system em- ployed for these measurements, K, was again found to be 7.0 f 0.5 X 10e4 mole liter-l at pH 8.0. Since this value of K, was sufficient to yield a straight line plot above pH 6.5 when employed with the data from these experiments in Equation 1, it appeared justifiable on the basis of the ex- perience with ATrEE just outlined to determine k, directly from the initial slope of this type of plot. This is further strengthened by consideration of the KS/K1 ratios calculated for ATrNH2, 0.5, and ATNH2, 0.3 at pH 7.9 (3). As a result of several measurements of kS of ATEE in the pH region 8 to 9, the maximal value of Jcs for this substrate was set at 2.87 f 0.10 mole liter-l min.-l mg. N ml.-‘. On this basis the values obtained in this pH-dependence series are shown in Fig. 6. In this case the theoretical line required a pK’ of 6.74 for the best fit of the data. This is close to the value of 6.70 determined for ATrEE, the difference presumably being due to small errors in setting limits for the theoretical lines and to the expected experimental error.

Comparison of Other Chymotrypsins-Since two other forms of chymo- trypsin, /3-chymotrypsin and y-chymotrypsin, have been isolated from the supernatant solution of crystalline a-chymotrypsin preparations (lo), it appeared of importance to characterize the pH-dependence of these en- zymes. A series of runs at varying pH values was made with each enzyme with ATrEE as substrate. The data were plotted according to Equation 3. Since previous investigators (10) have found little or no evidence that the kinetic constants of these enzymes differ from those of the QI form, the value of K, obtained for ATrEE with cr-chymotrypsin was used to calcu- late KS/K, for these enzymes. The data are shown in Fig. 4. It may be seen that Kr varies in approximately the same way for all three forms of chymotrypsin. Values of k~ were calculated from the intercepts as before and plotted as per cent of maximal lcs in Fig. 7. The pK’ value producing the best fit to the experimental data was 6.75 for r-chymotrypsin and 6.67 for ,&chymotrypsin. The maximal value of k, for y-chymotrypsin was

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294 a-CHYMOTRYPSIN-CATALYZED ESTEROLYSIS

determined to be 0.78 f 0.04 mole liter-l min.-’ mg. of N-l ml., which is identical within experimental error with that obtained for or-chymo- trypsin. For P-chymotrypsin a value of 0.53 f 0.03 mole liter-l min.-l mg. of N-l ml. was obtained for kB maximum. Unfortunately, this prep- aration appeared to contain amorphous material which could not be re- moved easily by crystallization. Thus, the low value obtained for kg is

2 IOO- . l *a.

2

+ 60

zl

u

[II 20

if2 f, /,

6 7 0 9 6 7 8 9 (a) P" lb)

FIG. 7. The dependence of the degree of esterolytic activity of p- and r-chymo- trypsin upon pH. Substrate, ATrEE; T 25”. a, &chymotrypsin; b, y-chymotrypsin.

TABLE I

Summa,ry of pH-Dependence Experiments

ol-cat

‘I

“

&Cht

y-Cht

Substrate

ATrEE “

ATEE ATrEE

1‘

K,

“C. mole L-1 mole L-1 min.-l mole 1.-l min.-l mg. N-1 ml. mg. N-1 ml.

25 6.70 f 0.03 8.9 f 2 X 1O-5 0.78 f 0.030.76 f 0.04

14 7.00 f 0.05 (9 x IO--6)J 0.38 f 0.020.36 f 0.03 25 6.74 f 0.05 7.0 f 0.5 X 1OP 2.87 f 0.102.78 f 0.1 25 6.67 f 0.05 (9 X 10-a)$ 0.53 f 0.030.51 f 0.04

25 6.75 f 0.05 (9 X lO+)$ 0.78 zk 0.040.76 f 0.05

* The average value of ka between pH 8.3 and 8.5. t Cht is used as a contraction for chymotrypsin. f Assumed from the value for a-chymotrypsin and ATrEE at 25”.

probably due to the presence of inactive protein. The comparison of rates of change of activity with pH remains valid, however, since it is made on the basis of per cent of maximal k, for each enzyme. The various values determined for the pK’ producing the best fit to the experimental data are collected in Table I. It may be seen that a value of 6.71 f 0.05 would satisfactorily describe all the data, within the limits of experimental varia- tion, except for the experiment carried out at 14”.

Comparison of cu-Chymotrypsin Preparations-The question of the vari- ability of samples of cY-chymotrypsin prepared according to various pro-

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L. W. CUNNINGHAM AND ‘2. S. BROWN 295

cedures or obtained commercially has been a matter of some concern. To test the reliability of enzyme preparations obtained by different meth- ods, a comparison was made between the crystalline cu-chymotrypsin used in all the other experiments, the same preparation after two alcohol re- crystallizations (17), and a commercially obtained alcohol recrystallized preparation. Table II shows the comparison of these samples at pH 8. It is apparent that the commercial preparation is only about 92 per cent as active as the other preparations. Such agreement appears remarkable in view of our current understanding of the complex nature of the chymo- trypsinogen activation process (18).

TABLE II

Comparison of a-Chymotrupsin Preparations

Enzyme Substrate

Salt fractionation (Kunitz) ATrEE ATEE

Same + 2 alcohol recrystallizations, . ATrEE ATEE

Commercial alcohol recrystallized.. ATrEE ATEE

ks

mole z.-$ni$ mg.

0.72 2.67

0.72 2.70 0.66

2.50

DISCUSSION

The chymotrypsin substrate employed in the majority of investigations reported here was ATrEE. As this compound exists in solution in only one form over the pH region investigated, any pH-dependent variation ob- served in this enzyme substrate system must be ascribed to changes in the enzyme. The excellent agreement obtained between experimental data describing the variation in ka for the cr-chymotrypsin-catalyzed hydrolysis of ATrEE with pH and a theoretical titration curve would seem to justify two conclusions. The first is that the loss of a proton from some weakly acidic group in the enzyme, pK’ 6.7, is necessary before enzymatic activity is det,ectable. It seems reasonable to assume that k3 as defined in the cur- rent formulation of the Michaelis-Menten theory (2) for this system ac- tually does not change. Rather, the change in the rate of the reaction with pH is due to the variation of the amount of the enzyme present in the catalytically active form. This is further substantiated by the close agreement of the activity versus the pH curve for another substrate, ATEE, with that obtained for ATrEE.

The second conclusion we may draw from this agreement is that, within

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the limits of these determinations, there is no evidence for heterogeneity of the enzyme preparation. Thus, whatever the cause of the heterogene- ous appearance of ar-chymotrypsin in electrophoresis (S), it appears that the ionization constant of a functional group critical for enzymatic activity is the same for all of the components. This interpretation is supported by the results obtained with /3- and y-chymotrypsin. Within the limits of measurement these two forms of chymotrypsin are kinetically identical to the a! form. Chemical studies of these proteins have also indicated a close similarity to the a! form of the enzyme. The N- and C-terminal resi- dues are all identical, but the P and y forms yield a greater variety of frac- tional amounts of amino acid per mole of protein when treated with car- boxypeptidase (18). Thus, an unexplained difference in solubility of the various preparations remains the chief distinguishing characteristic (10). Whatever the structural changes involved, it is apparent that they do not affect, and thus presumably do not involve, the portion of the molecule concerned with the specific catalytic activity.

The shift in the pH-activity curve which was obtained on lowering the temperature is characteristic of the acidic groups in proteins known to ionize in this pH region (19). By utilizing the values for -log K’ at 14”, 7.00, and at 25”, 6.70, for the reaction between or-chymotrypsin and ATrEE, a value for AH, the heat of ionization, for this functional group associated with enzymatic activity may be calculated to be approximately 11 kilo- calorie mole-l. Gutfreund (9), working with the trypsin-benzoyl-n-argi- nine-ethyl ester system, has determined the pK’ for trypsin4 to be 6.25 at 25” and the heat of ionization to be 7 kilocalorie mole-l. The agree- ment of these values with those reported for imidazole groups in proteins (19) has led him to suggest that a histidine residue in the trypsin molecule is directly concerned with the catalytic activity of the protein. In a-chy- motrypsin the pK’ associated with enzymatic activity also lies within the range associated with the imidazole group, but the value determined for AH more closely corresponds to those determined for amino groups. How- ever, it appears unlikely that terminal amino groups have pK’ values be- low 7.4 (19). Chemical analysis of the protein (10) has failed to provide evidence for any type of non-amino acid prosthetic group which might give rise to the observed variation with pH. Obviously any postulates based on these facts overlook the possibility that the specific spatial location of the group in the enzyme molecule may have greatly changed its acidic character from that usually encountered. Further speculations as to the nature of this group or groups in these closely related proteolytic enzymes must therefore await the availability of other types of information.

4 In a brief study of this system we have determined the pK’ for trypsin activity to be about 6.40. Our system, however, contained 0.10 M CaClz to stabilize the enzyme.

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The variation of K, for ATr with pH must also be considered in a dis- cussion of the nature of the ionizable group necessary for enzymatic ac- tivity. The suggestion that there is a negatively charged group in or near the act,ive center of chymotrypsin was first made by Neurath and Schwert (2) on the basis of kinetic studies of anionic and neutral specific inhibitors. Recently, Foster and Niemann (5) reinvestigated this point, utilizing a series of more closely related inhibitors. In measurements at pH 6.9 and 7.9, they detected a variation in K, for anionic inhibitors such as ATr, but could find no significant change for neutral inhibitors such as acetyl- n-tryptophanamide. On the basis of these and other comparative studies, they concluded that in or near the active center of the enzyme there is at least one negatively charged group. This group is postulated to be chiefly in the conjugate acid form at pH 6.9 and much less so at 7.8. The re- sults we have obtained for K, for ATr in the esterase system are generally in accord with such a hypothesis. However, the scope of the determina- tions is insufficient to warrant any quantitative statement concerning the actual degree of protonation at any pH, except that it is apparently close to 100 per cent near pH 5.5. The pK’ for the ionization of ATr is far too low to consider the variation of the COO/COOH ratio of this compound as a major factor in the pH-dependence of the affinity of the enzyme for ATr in the region above pH 5.

,4 similar variation in the degree of binding was found by Doherty and Vaslow (20) in their study of the thermodynamics of the interaction be- tween acetyl-3,5,-dibromo-L-tyrosine and chymotrypsin. These workers mere considering this compound as a substrate for the enzyme, whereas in our measurements the interaction between ATr and the enzyme was measured as competitive inhibition. It seems probable that the physio- logical hydrolytic function of this enzyme is dependent on the fact that the interaction between the protein- and carboxyl-substituted substrates re- mains constant as the pH is increased toward the optimum, while the interaction between enzyme- and carboxyl-unsubstituted “substrates” declines. The nature of the negatively charged group in the active center is unknown. Its relationship to the group of pK’ 6.7 which controls the catalytic function of the enzyme is likewise uncertain.

It may be seen that all three chymotrypsins are approximately 95 per cent in the cat,alytically active form at pH 8 and that the rate of increase with pH toward 100 per cent is small between pH 8 and 9. Extensive investigations were not carried out above pH 9 owing to the uncertainties introduced by the instability of the enzyme and substrate and the titri- metric difficulties common to the high pH region. The presence of high concentrations of calcium ions further contributed to the decreasing ef- fectiveness of the esterase method at high pH. In a few exploratory ex- periments, however, some evidence was obtained which indicated that the

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apparent K, of the reaction begins to increase rapidly above pH 9. It is difficult, therefore, to set a pH optimum for the enzymes and the two ester substrates which were used. For operational purposes the pH optimum is probably best defined in this case as the pH region from S to 8.5, where the apparent k3 is from 95 to 98 per cent of the maximal kS. In this region the cw-chymotrypsin-catalyzed hydrolysis of ATrEE approaches zero order kinetics at substrate concentrations below 0.01 M. Under these conditions, therefore, this substrate would appear to be nearly ideal for routine assay

of or-chymotrypsin.

The assistance of Dr. James E. Hurley and Mr. E. K. Carney in the per- formance of some of the initial measurements is gratefully acknowledged.

SUMMARY

The action of (Y-, p-, and r-chymotrypsin preparations on two synthetic substrates, ATrEE and ATEE, has been studied as a function of pH. In all cases the variation of the apparent JG3 with pH could be described by the assumption that enzymatic activity of the protein required the loss of a proton from a particular weakly acidic group in the protein. Within the limits of the experiment, the pK’ of this group was found to be the same, 6.71 =t 0.05, in all cases examined. While no evidence could be obtained from these studies which might indicate heterogeneity of the enzyme preparations, the close similarity of behavior of the three forms of chymotrypsin demonstrates the limited sensitivity of this test. Meas- urement of the change in pK’ with temperature led to a value of AH of ionization of 11 f 2 kilocalorie mole-i. The relationship of these values to those which have been determined for known functional groups in pro- teins has been discussed.

It has been shown that the apparent KI for one of the products of the reaction, ATr, is a continuous function of the pH of the system over the range pH 5.5 to 8. Possible explanations for this behavior have been dis- cussed.

BIBLIOGRAPHY

1. Cunningham, 1,. W., Carney, E. K., and Hurley, J. E., Federation Proc., 14, 199 (1955).

2. Neurath, H., and Schwert, G. W., Chem. Rev., 46, 69 (1950). 3. Foster, R. J., Shine, H. J., and Niemann, C., J. Am. Chem. Sot., 77, 2378 (1955).

4. Foster, R. J., and Niemann, C., J. Am. Chem. Sot., 77, 1886 (1955). 5. Foster, R. J., and Niemann, C., J. Am. Chem. Sot., 77, 3365 (1955). 6. Foster, R. J., and Niemann, C., Proc. Nat. Acad. SC., 39, 999 (1953).

7. Cunningham, L. W., J. Biol. Chem., 207,443 (1954). 8. Egan, R., Federation Proc., 12,199 (1953). 9. Gutfreund, H., Tr. Faraday Sot., 61,441 (1955).

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Leon W. Cunningham and Charles S. BrownESTEROLYSIS

-CHYMOTRYPSIN-CATALYZEDαKINETIC CONSTANTS OF

THE INFLUENCE OF pH ON THE

1956, 221:287-300.J. Biol. Chem. 

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