THE JOURNAL OF B~LOGKAL CHEMISTRY Vol. 246, No. 9, Issue of May 10, pp. 2918-2925, 1971

Printed zn U.S.A.

Investigations of the Chymotrypsin-catalyzed

Hydrolysis of Specific Substrates

V. DETERi\lI?;ATIO?; OF PRE-STEADY STATE KINETIC PARAMETERS FOR SPECIFIC SUBSTRATE ESTERS BY STOPPED FLOW TECHNIQUES*

(Received for publication, June 5, 1970)

JAMES MCCOXN,~ EDMOND Ku,f ALBERT HIMOE:$ KARL G. BRANDT,~ AND GEORGE P. HESS& 0

From the Section of Biochemistry and Molecular Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14850

SUMMARY

It has been suggested that the chymotrypsin-catalyzed hydrolysis of specific substrates is described by a three-step mechanism:

KS r” E + S = ES - EPz - Pz,

kz3 Axa

and that for esters k23 is much greater than ka4, and Kts is greater than the steady state kinetic parameter K,(app). Steady state kinetic investigations do not allow one to deter- mine the individual parameters for the mechanism shown above. This information can be obtained from pre-steady state kinetic investigations.

In this paper we are reporting pre-steady state kinetic parameters, determined by stopped flow techniques, for the chyrnotrypsin-catalyzed hydrolyses of the ethyl esters of N-acetyl-L-tyrosine, together with previously published data for the ethyl esters of N-acetyl-L-tryptophan and N-acetyl- L-phenylalanine. This allows a comparison of K’,, k23, and ka4 for the catalyzed hydrolyses of the esters of all three aromatic amino acids which are considered to be specific for chymotrypsin. Also included is an investigation of the pre- steady state kinetic parameters pertaining to the hydrolyses of N-acetyl-L-leucine methyl ester and of the methyl ester and amide of tosyl-L-arginine. N-Acetyl-L-leucine methyl ester was chosen because it allows an investigation of the pH dependence of kz, a study that is not possible with esters of aromatic acids, since in these reactions kz3 becomes too large above pH 6 to be adequately measured by the stopped flow

* This research was supported by grants from the National In- stitutes of Health and the Sational Science Foundation.

1 National Institutes of Health Postdoctoral Fellows. Present addresses of Professors McCann, Himoe, Brandt, and Hess are, respectively, Department of Biochemistry and Biophysics, Uni- versity of Hawaii, Honolulu, Hawaii 96822; Department of Bio- chemistry, Bavlor Universitv College of Medicine, Houston. Texas 77025; Dkpartment of Biochemistry, Purdue University, Lafay- ette. Indiana 47907: and MRC Laboratorv of Molecular Bioloev. Cambridge, England (1969 to 1970), Co&e11 University, Ithaca: New York 14850 (1971).

$ To whom reprint requests should be addressed at 210 Savage Hall, Cornell University, Ithaca, New York 14850.

technique. The pH dependence of kta was found to be simi- lar to the pH dependence of ka4. This information has not been available previously. p-Tosyl-L-arginine methyl ester and N-cr-p-tosyl-L-argininamide were included because, as typical trypsin substrates that are hydrolyzed by chymotryp- sin also, they are expected to offer some insight into the specificity of the reaction. Unlike in the chymotrypsin- catalyzed hydrolysis of specific substrate esters, the accumu- lation of an intermediate, such as EP2 in the above equation, could not be detected. One obvious explanation for this observation is that k23 rather than ka4 is rate-limiting. It is shown that unproductive binding of the unspecific substrate can also account for the data.

In preceding papers of the present series (l-6), we have reported kinetic as well as equilibrium measurements of the chymotrypsin-catalyzed hydrolysis of some specific substrate esters and amides at selected pH values. A variety of methods, including stopped flow measurements of the displacement of the dye proflavin from enzyme by substrate, was used to detect intermediates in the reaction and to measure pre-equilibrium parameters. In the preceding paper of this series (6), we showed that the proflavin displacement method gives the same infor- mation as direct measurements of products of the reaction.

The results of our investigations indicated that the chymo- trypsin-catalyzed reactions which we studied can be described at neutral pH and 25” in terms of the equation

PI % ha/1 k

E+S’ (1)

ES - EP2 -=E+P,

where is represents substrate ester or amide, ES an enzyme- substrate complex, EP2 an enzyme-substrate compound, PI . an alcohol or amine, and Pz a free acid. At higher temperatures or pH values, the formation of enzyme-substrate complexes involves at least two different conformations of the enzyme, and the pH-dependent equilibrium between these enzyme conformations accounts for the pH dependence of the catalytic reaction at alkaline pH (1).

2918

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Issue of May 10, 1971 J. &IcConn, E. Ku, A. Himoe, K. G. Rrandt, and G. P. Hess 2919

The mechanism shown in Equation 1 was first proposed (7, 8) for the chymotrypsin-catalyzed hydrolysis of a model substrate, p-nitrophenyl acetate, although in this particular reaction there is no evidence (9) for an enzyme-substrate complex. Steady state kinetic investigations, which for the mechanism shown in Equation 1 can yield only complex kinetic constants, indicated to Bender et al. (10, 11) that the mechanism holds for the chymotrypsin-catalyzed hydrolysis of the ethyl esters of acetyl-nn-tryptophan and acetyl-nn-phenylalanine, but not for carbobenzoxy-L-tyrosine ethyl ester.

In this paper we are reporting pre-steady state kinetic param- eters, determined by stopped flow techniques, for the chymo- trypsin-catalyzed hydrolyses of the ethyl esters of tyrosine (Ac-Tyr-0Et)r at pH 5.0, together with previously published data for the ethyl esters of tryptophan (Ac-Trp-OEt) and phenylalanine (Ac-Phe-OEt). This allows a comparison of K’s, k23, and k34 (Equation 1) for the catalyzed hydrolyses of the esters of all three aromatic amino acids which are considered to be specific for chymotrypsin. Also included is an investi- gation of the pre-steady state kinetic parameters pertaining to the hydrolyses of acetyl-n-leucine methyl ester (Ac-Leu-OMe) and of the methyl ester and amide of tosyl-n-arginine (Tos-Arg- OMe and Tos-Arg-NH,). Ac-Leu-OMe was chosen because it allows an investigation of the pH dependence of kZ3 (Equation 1)) a study that is not possible with esters of aromatic amino acids, since in these reactions kZ3 becomes too large above pH 6 to be adequately measured by the stopped flow technique. Tos- Arg-OMe and Tos-Arg-NH, were included because, as typical trypsin substrates that are hydrolyzed by chymotrypsin also, they are expected to offer some insight into the specificity of the reaction.

EXPERIMENTAL PROCEDURE

Three times crystallized, salt-free oc-chymotrypsin (Lots 6164, 6148-9, 6127-8, 5KB, 6JF, 6LD, and 7CD) was obtained from Worthington. &Chymotrypsin was prepared just before each use by activation of chymotrypsinogen (crystalline, Worthington) with trypsin (twice crystallized, Worthington), under conditions known to yield essentially the 6 form of the enzyme (12, 13). A molecular weight of 25,000 was assumed for the enzymes (14). Concentration of active enzyme was determined by the N-trans.cinnamoyl imidazole method (15). Protein concentration of enzyme solutions was determined spectrophotometrically at 280 mp, with the use of a molar extinc- tion coefficient of 50,000 M-I cm-l (16).

Ac-Phe-OEt (Lot G 2441, with melting point of 150”) p-tosyl- n-arginine (Lot S 1080, homogeneous by paper chromatography) and proflavin sulfate (Lot N 2200) were obtained from Mann. After recrystallization from water-methanol (17), the pro- flavin sulfate had a molar extinction coefficient at 444 rnp of 37,900 M-I cm-r. Ac-Tyr-OEt (Lot R-3669R), with melting point of 52-60” and [@Ii4 = +21” (c, 2, in ethanol), was ob- tained from Cycle. Ac-Leu-OMe (Lots K-5072 and K-5831)

1 The abbreviations used are : Ac-Tyr-OEt, N-acetyl-L-tyrosine ethyl ester; Ac-Phe-OEt, N-acetyl-n-phenylalanine ethyl ester; Ac-Trp-OEt, N-acetyl-L-tryptophan ethyl ester; Ac-Leu-OMe, N-acetyl-L-leucine methyl ester; Tos-Arg-OMe, p-tosyl-r-arginine methyl ester; Tos-Arg-NHz, AJ-ol-p-tosyl-n-argininamide; Tos- Arg, p-tosyl-n-arginine; Ac-Trp-NH*, N-acetyl-n-tryptophanam- ide; Ac-Phe-NHS, N-acetyl-r-phenylalaninamide.

was also obtained from Cycle; after purification as previously described (18), it had a melting point of 43.0”, in agreement with the previously reported value (18). A11 reported melting points are uncorrected.

All other materials were reagent grade and obtained from Mallinckrodt.

Apparatus

A Cary model 14 recording spectrophotometer with 10.mm silica cells and 0 to 2 or 0 to 0.2 slide wires was used for the spec- trophotometric measurements.

For the stopped flow experiments, a Gibson-Durrum stopped flow spectrophotometer was used. This instrument has cells with light path of 20 or 5 mm, and a tungsten-iodide light source with grating monochromator. Time-dependent change in light transmission of the experimental solutions was recorded on a Tektronix 564 storage oscilloscope or on a Tektronix 545B oscilloscope. “Dead time” of the instrument with 20-mm light path cell, determined as previously described (19), from meas- urements of ascorbic acid-potassium ferricyanide systems, was found to be about 4 msec.

A Radiometer pH-stat was used for steady state kinetic meas- urements of ester hydrolysis.

Methods

Stopped Flow Experiments-Measurements of kZ3, k34, and K’, (Equation 1) were made by the proflavin displacement method described previously (1, 5). Experiments with Ac-Phe- OEt, Ac-Tyr-OEt, and Tos-Arg-OMe as substrate were per- formed at pH 5.0 and at temperatures of 25.5”, 25”, and 26”, respectively. Experiments with Ac-Leu-OMe as substrate were performed at nine pH levels in the range 5.1 to 8.0, at both 15” and 25”.

For all of the measurements, solutions containing ol-chymo- trypsin and proflavin in the appropriate 0.1 M buffer solution (potassium acetate or sodium or potassium phosphate or pyro- phosphate, prepared as described in Biochemists’ Handbook (20), were mixed in the stopped flow apparatus with solutions of equal volume containing substrate in the same buffer. All solutions contained sufficient KC1 to give an ionic strength of 0.4. Initial concentrations in the Ac-Phe-OEt, Ac-Tyr-OEt, and Tos-Arg-OMe experiments were 10 pM enzyme, 50 PM

proflavin, and substrate in the ranges of 0.95 to 9.5 mM (Ac-Phe- OEt), 5 to 20 mM (Ac-Tyr-OEt), and 0.06 to 2.75 mM (Tos-Arg- OMe). Initial concentrations in the Ac-Leu-OMe experiments were 10 ,UM enzyme, 60 PM proflavin, and substrate in the range of 2.5 to 80 mM. The time-dependent change in concentration of the enzyme-proflavin complex was followed at 465 rnp. An observation cell with a 5-mm light path was used for all experi- ments except those with Ac-Leu-OMe and Tos-Arg-OMe, for which a 20.mm light path cell was used. Calculations were made as described under “Results.”

Steady State Kinetic iieasurements-Hydrolysis rates of the esters Ac-Tyr-OEt, Ac-Phe-OEt, Tos-Arg-OMe, and Ac-Leu- OMe were measured by continuous titration of the liberated acid on the pH-stat. In all cases, initial rates were determined at several substrate concentrations. The steady state kinetic constants kcat and K,(app) were calculated by means of a digital computer program written for the Lineweaver-Burk form of the Michaelis-Menten rate equation (21). Data weight-

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2920 Chymotrypsin-catalyzed Hydrolysis of Speci$c &&rates. V Vol. 246, No. 9

ing and the calculation of standard errors of the kinetic constants were performed as discussed by Wilkinson (22).

In the experiments with Ac-Tyr-OEt and Ac-Phe-OEt, the measurements were performed at pH 5.0 and 25.5” in 0.4 M

KCl; initial enzyme concentration was -1 pM, and initial sub- strate concentration varied in the ranges of 0.2 to 5.0 mM Ac- Tyr-OEt and 0.2 to 7.0 mM Ac-Phe-OEt. In the case of Tos- Arg-OMe, the experiments were performed at pH 6.7 and 25” in 0.4 M KCl; initial enzyme concentration was ~60 PM, and initial substrate concentration varied in the range of 0.4 to 0.12 mnd. In the case of Ac-Leu-OMe, the experiments were performed at pH 6.0 and 25” in 0.4 M KCI; initial concentrations were ~1 pM enzyme, and substrate in the range of 0.6 to 39 mM.

Spectrophotometric Determination of Chymotrypsin-Substrate Dissociation Constants-Values of K’s pertaining to Tos-Arg-OMe at pH levels 6.65 and 5.0, Tos-Arg-NH2 at pH 5.0, and Tos-Arg at pH 6.8 were determined with the use of the proflavin-dis- placement method and calculated as previously described (5). The determination is based on absorbance of the enzyme-pro- flavin complex, and utilizes absorbance difference measurements, made on the Cary spectrophotometer, of mixtures of enzyme, substrate, and proflavin solutions. In most experiments, the solutions were measured at 500 ml.c, at which there is practically no absorbance due to proflavin, and at 465 ml.c, at which absorb- ance due to the enzyme-proflavin complex is maximal. In a few experiments, the measurements were made at 465 and 440 mp. The experimental technique was adapted as required so as to yield a positive absorbance difference measurement, AA:!“,-,,, or AA%,40, which was taken to be a measure of the amount of enzyme-proflavin complex present in the experimental solution. Measurements were made at room temperature, about 25”, with variation less than 0.3” during the period of observation. Readings were completed within 3 to 4 min after mixing of the particular stock solutions used in each experiment; during this time only a negligible amount ( <50/,) of substrate hydrolyzes, as shown by calculation from steady state kinetic parameters of these systems.

The experimental solutions were prepared from stock solu- tions of the reactants and buffer. The proflavin sulfate stock solutions were prepared fresh daily in boiled water which had been cooled under nitrogen, and were kept protected from the

a Time -

1. Photographs of oscilloscope traces of transmittance at 465 w in a stopped flow experiment with Ac-Leu-OMe at pH 8.0 and 25” (see Table I). The time scale in the first part of the ex- periment is 0.05 set per cm; for the other part shown it is 5 set per cm. Recording of the initial fast increase in transmittance, not shown, would have required a third time scale. Initial solution concentrations were 1.4 mu Ac-Leu-OMe, 60 PM or-chymotrypsin, and 60 PM proflavin in 0.1 M pyrophosphate buffer; ionic strength = 0.4 (with KCl).

light. Enzyme stock solutions were prepared either in milli- molar HCl or in the appropriate buffer, and substrate stock solutions were prepared in buffer. For the experiments with Tos-Arg-OMe at pH 6.65, initial concentrations in the experi- mental solutions were 20 pM enzyme, 20 pM proflavin, and substrate in the range of 0.22 to 1.0 mM; solutions contained phosphate buffer to yield an ionic strength of 0.1. The experi- mental solutions for the measurements at pH 5.0 were buffered with 0.1 M acetate, and contained sufficient KC1 to give an ionic strength of 0.4. Two sets of experiments were performed with Tos-Arg-OMe at pH 5.0: initial concentrations were (a) 39 PM enzyme, 40 FM proflavin, and substrate in the range of 0.05 to 0.62 mM, and (b) 41 pM enzyme, 40 pM proflavin, and substrate in the range of 0.34 to 1.03 mM. In the Tos-Arg-NH, experi- ments at pH 5.0, initial concentrations were 41 pM enzyme, 40 PM proflavin, and substrate in the range of 0.27 to 4.2 mM. For the experiment with Tos-Arg at pH 6.8, initial concentrations were 28 PM enzyme, 20 pM proflavin, and substrate in the range of 0.30 to 0.59 mM; solutions were buffered with phosphate to give an ionic strength of 0.1.

In a given experiment, the dissociation constant of the enzyme- substrate complex, K’S, was calculated from the observed ab- sorbance difference measurements of the ternary mixtures at various substrate concentrations. The calculation, which requires also a separate determination of the dissociation constant for the enzyme-proflavin complex, KBF, was made by means of a computer program, as described previously (5). Values of Kgp used in calculating the results of the experiments reported here are 39 PM at pH 5.0, 44 pM at pH 6.65, and 25 JLM at pH 6.8. Measurement of KBF also yields A~MBF, the molar extinction coefficient of the enzyme-proflavin complex. Measurement of KBF also yields A@9 the molar extinction coefficient of the en- zyme-proflavin complex. Although values of Ae~fiF were found to be about 20% higher for the AA:!&,,, measurements than for the AA,0!&,,, measurements, comparable values were obtained for the dissociation constants.

RESULTS

Stopped Flow Measurements of Chymotrypsin-catalyzed Hy- drolysis of Substrate Esters-A typical oscilloscope trace ob- tained when excess ester substrate is mixed with enzyme and proflavin in the Gibson-Durrum spectrophotometric stopped flow apparatus is shown in Fig. 1. This trace was obtained at pH 8.0, 25”, and 465 ml.c, with Ac-Leu-OMe as substrate. Initial concentrations in the experimental solutions were 1.4 mM Ac- Leu-OMe, 6 PM enzyme, and 60 pM proflavin; the solution was buffered with phosphate to give an ionic strength of 0.4. Inter- pretation of a trace such as this is made on the basis of the following observations. The formation of a chymotrypsin- proflavin complex results in a perturbation, with a maximum at 465 rnp, of the proflavin absorption spectrum. Introduction of substrate into a system of enzyme and proflavin results in competition between proflavin and substrate for the enzyme; consequently, less proflavin is bound by the enzyme, and the absorption due to complex is decreased. The observable re- action steps are therefore interpreted as follows. (a) An initial rapid decrease in concentration of the enzyme-proflavin com- plex, EF, considered to reflect the formation of an enzyme-sub- strate complex (ES in Equation 1). This change is usually too fast to be measured (it is not shown in Fig. 1). (b) A second decrease in the concentration of EF, considered to occur as a

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Issue of May 10, 1971 J. McCann, E. Ku, A. Himoe, K. G. Brandt, and G. P. Hess 2921

result of the formation of another intermediate, such as EP2 in Equation 1. This rapid but measurable step has an observed rate that is dependent on both pH and substrate concentration. (c) A period during which essentially no change in EF occurs- a period considered to reflect the time during which there is maintained a steady state concentration of the intermediate such as EP2 in Equation 1. The length of this period depends on substrate concentration. (d) Finally, an increase in the con- centration of EF, considered to be a result of the decomposition of EP2 and regeneration of free enzyme.

When measurements such as the one shown in Fig. 1 are made as a function of substrate concentration, the constants lc23, k34, and K’s (in Equation 1) pertaining to the chymotrypsin-cata- lyzed hydrolysis of substrate esters in the presence of proflavin can be evaluated by use of the equation

k kzaS0

Ohs = so + Kill + (FO/KEF)I + k34 (2)

which was derived for the conditions SO >> E. (5). The other assumptions under which Equation 2 is valid have been stated also (5). In this equation, /cobs is the observed rate constant for the attainment of a steady state, and k23, l&4, and K’, are parameters defined in Equation 1. Eo, So, and F. are initial analytical concentrations of enzyme, substrate, and proflavin, and KEF is the enzyme-proflavin dissociation constant.

In the case of Ac-Tyr-OEt, the kobs values obtained in the stopped flow experiments were considerably higher than values of the steady state kinetic parameter, kcat. Under these cir- cumstances, koat can be taken as a measure of ks4. This can be

90

(kobs-k34)(so)-‘Xlo-3 (M-‘set-‘1

FIG. 2. Stopped flow data obtained by the proflavin displace- ment method for the or-chymotrypsin-catalyzed hydrolysis of Ac-Tyr-OEt at pH 5.0 and 25”. Values of (k&s - k& are plotted against (k&s - kad)/So (see Equation 2 of the text). Values of ka4 were taken as equivalent to the steady state kinetic constant kcat (see text). In accordance with Equation 2, K’s can be ob- tained from the slope of the line and k23 from the intercept of the ordinate. Actual evaluations were made by computer according to the method of Wilkinson (21) (see values in Table II). Initial experimental solution concentrations were 5 to 20 mM Ac-Tyr-OEt, 10 PM a-chymotrypsin, and 50 MM proflavin in 0.1 M acetate buffer; ionic strength = 0.4 (with KCl).

seen from Equation 3, which defines kaat for the mechanism shown in Equation 1.

(3)

Rearrangement of Equation 2 allows evaluation of the pertinent parameters from a plot of (ICobs - k34) against (kobs - k3*)/&.

Such a plot for the chymotrypsin-catalyzed hydrolysis of Ac- Tyr-OEt at pH 5.0 and 25” is shown in Fig. 2. The intercept of the ordinate gives a k23 value of 83 see-l, and the slope gives a value for K’& + (FO/KBF)] of 38 mM. Actual evaluations were made by computer, according to the method of Wilkinson

(22). For the experiments with Ac-Phe-OEt and Ac-Leu-OMe, all

parameters were evaluated from plots of k&a against k&s/ SO. Such a plot-for the a-chymotrypsin-catalyzed hydrolysis of Ac-Phe-OEt at pH 5.0 and 25.5”-is shown in Fig. 3 (open circles). A digital computer program was devised to calculate the best i&z, k34, and K’, values for all of the kobs values ob- tained in a given experiment. The computed values are rep- resented by the slope and intercept of the solid line in Fig. 3; the triangles represent values of (&,s - k34)/&,. The intercept of the ordinate by the solid line gives a lc23 value of 13 see-1, and the slope gives a value for K’& + (Fo/KE~)] of 15.6 mM.

Stopped flow measurements of the chymotrypsin-catalyzed hydrolysis of Ac-Leu-OMe were made at nine pH levels in the range 5.1 to 7.9, and at both 15” and 25”. Results are shown in Fig. 4 and Table I. In the figure, values of k23 and lc34 at 25” are plotted against pH; different scales are used since kZ3 (left ordinate in Fig. 4) is about 10 times larger than k34 (the right

\

0

0

0

‘t 0 A 0

‘A 0

\ 0

AA \

A

I I I 1000 2000 3000

k /S,, (K’sec’)

FIG. 3. Stopped flow data obtained by the proflavin displace- ment method for the or-chymotrypsin-catalyzed hydrolysis of Ac-Phe-OET at pH 5.0 and 25.5”. 0, values of kobs plotted against kobs/& (see Equation 2). From these data, best values of k23, k34, and K’S were established by a computer program (see text). Values are listed in Table II. A, values of (kobs - k& plotted against (kobs - k&/So. Initial solution concentrations were 0.95 to 9.5 mM Ac-Phe-OEt, 10 PM or-chymotrypsin, and 50 m pro- flavin in 0.1 M acetate buffer; ionic strength = 0.4 (with KCl).

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2922 Chymotrypsin-catalyzed Hydrolysis of Specific Substrates. V Vol. 246, No. 9

100

go-

eo-

i-o-

- GO- F

; 50-

';I 40-

30-

20-

IO-

04.0

PH

FIG. 4. The pH dependence of individual rate constants in the or-chvmotrvnsin-catalvzed hvdrolvsis of Ac-Leu-OMe at 25”. Values of ic’,, and k~; were ealcul”ated according to a computer program (see text) from observed rates measured in stopped flow experiments by the proflavin-displacement method. l , k23; n , k34. Note that different scales are used in the two plots. The left ordinate pertains to k23, and the right ordinate to k34. The dashed line shows k34 data plott,ed on the same time scale as k23. Internretation of these curves is that both rate constants are con- trolled by the ionization state of a group of the enzyme with pK- (kinetic) -6.8. Values of the constants at the various DH levels are listed in Table I. Initial concentrations in the experimental solutions were 2.5 to 80 mu Ac-Leu-OMe, 10 PM oc-chymotrypsin, and 60 PM proflavin in the appropriate buffer (see text) of strength 0.1 M. Ionic strength = 0.4 (with KCl).

TABLE I

Rate and equilibrium constants pertaining to e-chymotrypsin- catalyzed hydrolysis of Ac-Leu-OMe

Determinations were made by stopped flow measurements of proflavin displacement. Initial concentrations were 10 PM en- zyme, 60 PM proflavin, and substrate in the range of 2.5 to 80 mM. Experimental solutions contained 0.1 M buffer, as described for each pH in Biochemists’ Handbook (20), and sufficient KC1 to bring the ionic strength to 0.4.

PH Temperature kza kaa K’,p

set-’ see-' ??a4

5.1 25.0” 3.2 f 0.4 0.2 93 f 11 15.0 0.4 f 0.08 0.06 22 f 6

5.4 25.0 3.6 f 0.8 0.2 72 f 19 15.0 1.6 f 0.4 0.12 18 f 8

5.8 25.0 15.5 f 2.2 1.0 68 f 10 15.0 2.8 f 0.7 0.15 22 f 7

6.0 25.0 10.6 f 1.4 1.7 24 f 4 15.0 4.5 f 0.7 0.44 29 f 5

6.4 25.0 31 f 6 2.2 37 f 9 15.0 7.5 f 1.4 0.80 13 f 4

6.8 25.0 56 f 14 2.4 38 f 11 15.0 27 f 5 1.4 20 f 10

7.2 25.0 80 z!z 5 6.9 46 f 3 15.0 25 f 7 1.9 25 f 8

7.5 25.0 131 f 31 5.9 80 f 19 15.0 64 f 7 3.3 83 f 8

8.0 25.0 102 f 10 9.0 64 f 6 15.0 63 f 10 5.1 90 f 13

a Calculated from the observed slope and corrected for proflavin binding: slope = K’s [l + FJKJJ,F)]. Average values for KIF of 44 PM at 25” and 37 go at 15” were assumed for all pH levels.

ordinate). It can be seen that the magnitudes of both rate

constants depend on the ionization of a group with pK(app) -6.8. An idea of the relative magnitudes of the two constants may be obtained by comparing the line with solid circles, which pertains to k23, with the dashed line, which represents the k34 measurements plotted on the same scale (the left ordinate) as the k23 measurements. Table I lists all of the measured values of k23, k34, and K’s for this substrate. It may be noticed that K’s is essentially pH-independent in the pH region 5 to 8. A similar pH independence is exhibited by the steady state kinetic parameter K,(app) in the chymotrypsin-catalyzed hydrolysis of amides (1, 3, 5); equilibrium and stopped flow experiments have indicated that in the chymotrypsin-catalyzed hydrolyses of Ac-Phe-NH2 and Ac-Trp-NH2, K,(app) is a measure of K’s (5).

A comparison of the pre-steady state kinetic parameters k23, k34, and K’s for the ethyl esters of three aromatic amino acid derivatives-acetyl-n-tryptophan, acetyl-n-phenylalanine, and acetyl-L-tyrosine-and for Ac-Leu-OMe, the methyl ester of acetyl-n-leucine-is given in Table II. The values at pH 5.0 were measured in stopped flow experiments, as were the param- eters pertaining to Ac-Leu-OMe at pH 8. In the case of the aromatic amino acid esters, values for kZ3 and k34 near pH 8 could not be obtained directly from measurements in this pH region because of the large magnitude of kQ3 and the time resolution of the stopped flow machine. Instead, they were calculated from the pH 5.0 values on the basis of observations made in the experi- ments with Ac-Leu-OMe: pH independence of K’s in the pH region 5 to 8, and dependence of kZ3 and k34 on the ionization of a group of the enzyme with pK(kinetic) -6.8. The values of these parameters near pH 8 represent the pH-independent values of the rate constants.

The data in Table II show that ks4 for all four esters is suf- ficiently smaller than k23 so that the steady state kinetic param- eter kcat is essentially a measure of k~. It has previously been observed (24) that, in the chymotrypsin-catalyzed hydrolysis of esters, kcat increases with increasing pH, reaching a maximum at pH 8.0: the Jc,,~-~H rate profile follows the ionization of a group of the enzyme with pK(kinetic) -7.0, in agreement with the pH dependence of k34 determined in stopped flow experi- ments (see Fig. 4).

Table II also lists previously determined values of Ii’, for the amides of acetyl-L-tryptophan and acetyl-n-phenylalanine (Ac-Trp-NH2 and Ac-Phe-NHZ), thereby permitting a com- parison of K’, values for esters and amides of the same aromatic

amino acid derivatives. The values are of comparable magni- tude: 2.3 mM for Ac-Trp-OEt and 4.7 mM for Ac-Trp-NH2 and 7.3 mM for Ac-Phe-OEt and 28 mM for Ac-Phe-NHz. Although the measurements were made at different pH levels, the com- parisons are valid on the basis of the observation that K’s for both esters and amides is pH-independent in the pH region 5 to 8. As discussed above, mea.surements of the chymo- trypsin-catalyzed hydrolysis of the ester Ac-Leu-OMe show such a pH independence for K’,, and it has been shown previously that, in the chymotrypsin-catalyzed hydrolysis of amides, K’s is pH-independent in this pH region (5).

Steady State Kinetic Measurements of Chymotrypsiwxtalyzed

Ester Hydrolyses-Values of the steady state kinetic parameters koat and K,(app), determined by computer analysis of initial hydrolysis rates measured by pH-stat titration, are given in Tables II and III.

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2923 Issue of May 10, 1971 J. McCann, E. Ku, A. Himoe, K. G. Bran& and G. P. Hess

TABLE II Rate and equilibrium constants pertaining to a-chymotrypsin-catalyzed hydrolysis of speci$c substrate esters

Stopped flow and steady state measurements of the Ac-Trp-OEt reaction were reported previously, as indicated. Stopped flow measurements of the Ac-Phe-OEt and Ac-Tyr-OEt reactions were made at pH 5.0 by the proflavin displacement method. Initial con- centrations were 10 PM enzyme, 50 PM proflavin, and substrate in the range of 0.95 to 9.5 my Ac-Phe-OEt and 5 to 20 mM Ac-Tyr-OEt. Ionic strength = 0.4. The Ac-Phe-OEt experiments were performed at 25.5”, and the Ac-Tyr-OEt experiments at 25”. Steady state kinetic measurements of these reactions were made by pH-stat titration at pH 5.0 and 25.5’ (see Table III). Stopped flow measure- ments of Ac-Leu-OMe hydrolysis were made by the proflavin displacement method at pH levels 5.1,6.0, and 8.0 and at 25” (see Table I). Steady state data were obtained at pH 6.0 and 25” (see Table III). Comparative data on the amides Ac-Trp-NH2 and Ac-Phe- NH, were obtained around pH 8, under conditions indicated by the footnotes.

-

Substrate

Ac-Trp-OEt

Ac-Phe-OEt

Ac-Tyr-OEt

Ac-Leu-OMe

Ac-Trp-NH?

Ac-Phe-NH*

PH

5.0 -8

5.0 -8

5.0 -8

5.1 8.0 7.9 8.0 7.0 8.0 8.0

kza

SW-’

35 f 9b 22ood

13 f 2 84W

83 f 24 53OW

3.2 f 0.4 102 f 10

0.05f

0.05”

T-

Stopped flow

ksr

see-1

0.84~ 52d

2.2 14W

3.1 195d

0.2 9.0

K’s”

mdb

2.3 f 0.6b

7.3 f 1.5

18 f 6

93 f 11 64 f 6

4.7 f 0.5@

28 f 8” 28 f 7g

Steady state

-

bat

set-’

0.84~ 51.6e

2.5 168”

3.1 1938

1.3

0.05’

Km @PP)

??a-

0.083~

1.3

0.8

4.2

4.2’

31”

kw/K’s koat/Km (~PP)

marl set-1 n&r’ set-’

15b 1oc

1.8 1.5

4.6 5.1

0.4 0.3

_-

a Calculated from observed slope = K’s [l + (Fe/K BF , with Fo = 50~~ and KzF = 44~~. )] * Data published previously (5). Measured at 28”. Substrate concentrations were in the range of 0.5 to 2.25 mM. c Data of Bender et al. (10). Measured at 25”. d Values at pH 8.0 were calculated from the pH 5.0 data with the assumption that rate constants increase as an ionizing group with

pK(app) of 6.8 ionizes. This assumption is based on the experimentally observed pH dependence of k23 and kaa in the cu-chymotrypsin- catalyzed hydrolysis of Ac-Leu-OMe (see Table I).

e Reported values for Ac-Trp-OEt at pH 8.25 (23), for Ac-Phe-OEt at pH 7.8 (24), and for Ac-Tyr-OEt at pH 8.5 (25). f Data obtained previously (3). The steady state parameters k 0at and Kn(app) were measured by the automatic amide hydrolysis

methods of Lenard et al. (26) at 25” and pH 7.9, with ionic strength = 0.2 and initial concentration of 2 to 16 mM substrate and 40 pM enzyme. As previously shown (3), the steady state kinetic parameters k oat and Km(app) in the chymotrypsin-catalyzed hydrolysis of these amides correspond to ,& and K’s of Equation 1.

c Data published previously (5). K’ s value was measured by the proflavin displacement method at 24” and pH 8.0, with ionic strength = 0.4. Initial concentrations were 1.5 to 10.5 mM substrate, 50 PM enzyme, and 40 PM proflavin.

h Data of Foster and Niemann (27). Measured at 25” and pH 7.0, with ionic strength = 0.10. i Data published previously (4). Measured by spectral changes at 290 rnp at 24” and pH 8.0, with ionic strength = 0.5 and initial

oncentrations of 6 to 34 m&r substrate and 40 PM enzyme. C

In the chymotrypsin-catalyzed hydrolysis of Tos-Arg-OMe, TABLE III there was evidence of substrate activation at high substrate concentrations, a phenomenon that has been reported pre- viously (28). The listed parameters, however, represent values obtained at low substrate concentrations where activation does not measurably affect the results.

Determination of Enzyme-Substrate Dissociation Constants for Complexes of ar-Chymotrypsin with Tosyl Argfnine and Deriva- tives-Values of K’s (Equation 1) perta.ining to the ur-chymo- trypsin-catalyzed hydrolysis of tosyl arginine ester and amide are given in Table IV. Both Tos-Arg-OMe and Tos-Arg-NH, were measured at pH 5.0, the ester was also measured at pH 6.65, and Tos-Arg was measured at pH 6.8. These values of K’e were obtained by means of the proflavin displacement method, described under “Experimental Procedure”.

It is to be noted from inspection of Tables III and IV that K’s obtained in the equilibrium experiments with Tos-Arg-OMe at pH 6.7 and 25” (K’s = 0.35 mM) agrees within experimental

Steady state kinetic measurements of a-chymotrypsin-catalyzed hydrolysis of substrate esters

Measurements were made by pH-stat titration of liberated acid. Initial enzyme concentrations were -1 PM for the Ac-Tyr- OEt, Ac-Phe-OEtt and Ac-Leu-OMe experiments, and 60 PM for the Tos-Arg-OMe measurements. Temperatures were 25.5’ for Ac-Tyr-OEt and Ac-Phe-OEt experiments, and 25.0” for the Tos- Arg-OMe and Ac-Leu-OMe experiments. Ionic strength = 0.4 (with KCl).

Substrate

Ac-Tyr-OEt . . . Ac-Phe-OEt . Tos-Arg-OMe.. Ac-Leu-OMe

Initial substrate

pH concen- tration

hat Km (~PP)

ntM St%-’ WiM

5.0 0.2-5.0 3.1 0.8 5.0 0.2-7.0 2.5 1.3 6.7 0.4-0.120.0022 f 0.00010.14 f 0.01 6.0 0.639 13 f 0.03 4.2 zk 0.2

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Chymotrypsin-catalyzed Hydrolysis of Speci$c Substrates. V Vol. 246, No. 9

TABLE IV Dissociation constants (K’s) for complexes of a-chymotrypsin with

tosyl arginine and derivatives Results were obtained spectrophotometrically by the proflavin

displacement method at 25” (see text). The pH 5.0 solutions con- tained 0.1 M acetate and sufficient KC1 to give an ionic strength of 0.4; the pH 6.65 and pH 6.8 solutions were buffered with phos- phate to give an ionic strength of 0.1. Initial concentrations of substrate were in great excess of enzyme and proflavin (see “Ex- perimental Procedure”).

Substrate PH K’S

m?d

Tos-Arg-OMe 6.65 0.3 f 0.1 5.0 0.3 f 0.1

Tos-Arg-NH* 5.0 1.4 f 0.3 Tos-Arg 6.8 2.4 f 0.3

-

error with the steady state kinetic parameter K,(app) measured for the catalytic hydrolysis of this substrate at pH 6.7 and 25’ (K,(app) = 0.14 mM). It may also be noted from inspection of Table IV that the K’s values for the amide and ester derivatives of Tos-Arg are within a factor of about 5 of each other, the ester binding better than the amide. A similar relationship holds be- tween the K’s values of the ester and amide derivatives of the aromatic amino acids; in the case of the derivatives of N-acetyl- n-phenylalanine, the ester binds about 4 times better than the amide (see Table II).

Attempts to detect time-dependent changes at 465 rnp in stopped flow proflavin displacement experiments with 10 /.LM

chymotrypsin, 50 PM proflavin, and 0.06 to 2.75 mM Tos-Arg- OMe at pH 5.0 and 26” yielded no measurable results.

DISCUSSION

The applicability of Equation 1 to the chymotrypsin-catalyzed hydrolysis of specific substrate esters has been proposed mainly on the basis of steady state kinetic experiments of Bender et al. (10, 11). The conditions required to produce an accumulation of EP% (Equation 1) have also been discussed (8, 10, 11) : i&a > i&4 and K’e > K,(app). Information about individual rate con- stants cannot be obtained by steady state kinetic investigation, but can be obtained by flow methods; whereas the stopped flow measurements yield values of both k~ and i&4 (Equation l), the steady state experiments yield measurements of only a combina- tion of rate and equilibrium constants.

The first direct proof that k23 > k~ and K’s > K,(app) in the chymotrypsin-catalyzed hydrolysis of specific substrate esters was obtained in measurements of the catalytic hydrolysis of Ac-Trp-OEt by flow techniques yielding individual rate and equilibrium constants (1). We have now shown (see Table II) that these relationships also pertain to the other chymotrypsin- specific substrate esters, Ac-Phe-OEt and Ac-Tyr-OEt, and to Ac-Leu-OMe, which are also catalytically hydrolyzed by chymo- trypsin.

The catalytic rate constant, koat, of chymotrypsin-catalyzed reactions depends on an ionizing group with pK -7 (11, 24). Crystallographic experiments of Blow and co-workers (29-31) led to the suggestion that this pK(app) -7 reflects the interac- tion of 3 amino acid residues in the active site of the enzyme (see Diagram 1).

The numbering of the amino acid residues is based on the se- quence of these residues in the enzyme as determined by Hartley (32) and by Keil and S&m and co-workers (33). It is this inter- action of the 3 amino acid residues which is considered (31) to have an important effect on the catalytic reaction by increasing the nucleophilicity of Ser-195 by a transfer of electrons from the buried negatively charged @-carboxy group of Asp-102 to the Ser-195 oxygen via His-57. One might expect that the transient acylation of the Ser-195 oxygen by substrate during the catalytic reaction would affect the pK of the system since the interaction between His-57 and Ser-195 is prevented by this step. It became therefore of interest to determine the pH dependence of the rate constant kt3 (see Equation 1): this information could not have been obtained from steady state kinetic investigations of the chymotrypsin-catalyzed hydrolysis of specific substrate esters or of Ac-Leu-OMe, since in these reactions the steady state kinetic parameter is a measure of kar only (see Table II). In the chymo- trypsin-catalyzed hydrolysis of Ac-Leu-OMe, k23 has the same pH dependence as k34: a group with pK (kinetic) of about 6.7 controls both rate constants (Table I, Fig. 4). Evidence that this is also true for the chymotrypsin-catalyzed hydrolysis of specific substrate esters comes from direct determinations of the pH dependence of i&3 and k~4 in the chymotrypsin-catalyzed hy- drolysis of Ac-Trp-OEt, determined in flow experiments in the pH region 4.3 to 6.0 (1, 5). Additional pertinent evidence for similar pH dependences of k23 and k34 in the chymotrypsin-cata- lyzed hydrolysis of specific substrate esters comes from combined results of steady state kinetic investigations (3, 11, 23-25) and the studies presented here. The steady state kinetic investiga- tions indicate that kcat for the chymotrypsin-catalyzed hydrolyses of Ac-Phe-OEt, Ac-Trp-OEt, and Ac-Tyr-OEt is controlled by an ionizing group with pK(kinetic) 6.8, while K,(app) is pH- independent in the region in which the group with pK(kinetic) of 6.8 ionizes. The data obtained in the studies reported here are presented in Table II and indicate that, for the mechanism of Equation 1, JCcst for the ester substrates investigated is a measure of kad, and that lc34 < LB. For the mechanism shown in Equa- tion 1, K,(app) = K’s k34/k23; therefore, the observed pH inde- pendence of K,(app) in a pH region where kza is pH-dependent requires that k23 and lc34 have the same pH dependence.

Unlike in the experiments with specific substrate esters, a steady state intermediate could not be detected by stopped flow techniques in the chymotrypsin-catalyzed hydrolysis of Tos-Arg- OMe at pH 6.7 and 25.5”. I f it is assumed that the mechanism shown in Equation 1 is applicable to this reaction, the lack of evidence for a steady state intermediate could indicate that lc23 is rate-limiting in this catalytic reaction. It is unlikely that the process characterized by k23 in Equation 1 is too fast to be de-

Asp-CO&H-N &> 7 KH

57 N+-H***O-SW+ Asp-CO&*H-N **OH-0-Ser+H+ 102 \ 195 102 195

DIAGRAM 1

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Issue of May 10, 1971 J. McCann, E. Ku, A. Himoe, K. G. Bran&, and G. P. Hess 2925

tected bv stopped flow technioues: the known dead time of our ” __ plex. There are, of course, other possible explanations, involving stopped flow apparatus indicates that k23 would have to be better more complex mechanisms than the one shown in Equation 1. than 200 see-l. I f kZ3 were greater than 200 set?, the steady state kinetic parameter kcat (with a value of 2 x lop3 see-r, as

REFERENCES

shown in Table III) would be a measure of k34 and, since the 1. HESS, G. P., MCCONN, J., Ku, E., AND MCCONKEY, G.? Trans.

mechanism in Equation 1 requires that K,(app) be equal to Rou. Sot. B. 267. 89 (1970).

K’, k34/kZ3, K,(app) would have to be less than 1 PM (see values 2. BRA~DT, K. G., AND Hiss, G. P., Biochem. Biophys. Res. Com-

mun., 22, 447 (1966). of lc34 and Kfs in Tables III and IV). Experimentally, however, 3. HIMOE, A., PARKS, P. C., AND HESS, G. P., J. Biol. Chem., 242,

it was found that K,(app) = 140 PM, which is of the order of the 919 (1967).

independently determined Kfs value (Table IV). Therefore, 4. HIMOE, A.,. BRANDT, K. G., AND HESS, G. P., J. BioZ. Chem.,

provided that the mechanism in Equation 1 applies, the experi- 242, 3963 (1967).

5. BRANDT, K. G., HIMOE, A., AND HESS, G. P., J. BioZ. Chem. ments indicate that in the chymotrypsin-catalyzed hydrolysis of 242, 3973 (1967).

Tos-Arg-OMe kZ3 is the rate-limiting step. This situation of i&3 6. HIMOE, A., BRANDT, K. G., DESA, R. J., AND HESS, G. P.,

being rate-limiting has been observed (4,5) in the chymotrypsin- J. Biol. Chem., 244, 3483 (1969).

catalyzed hydrolysis of amides, but is contrary to results that 7. HARTLEY, B. S., AND KILBY, &. A:, Biochem. J., 66,288 (1954).

have been obtained in stopped flow measurements of the hydroly- 8. GUTFREUND, H., AND STURTEVANT, J. M., Biochem. J., 63, 658

(1956) . sis of chymotrypsin specific substrate esters, for which the rate- 9. FALLER, L., AND STURTEVANT, J. M., J. Biol. Chem., 241, 4825

limiting constant was found to be k34 (see Table II). Inagami (1966).

and Sturtevant (34), who investigated the chymotrypsin-cata- 10. BENDER, M. L., CLEMENT, G. E., K~~zDY, F. J., AND HECIC,

lyzed hydrolysis of the trypsin substrate benzoyl-n-arginine H. D’A., J. Amer. Chem. Sot., 86, 3680 (1964).

11. BENDER, M. L., AND K~ZDY, F. J., Annu. Rev. Biochem., 34,49 methyl ester, have suggested the possibility that k23 in the chymo- (1965).

trypsin-catalyzed hydrolysis of this substrate is much smaller 12. BETTELHEIM, F. R., AND NEURATH, H., J. BioZ. Chem., 212,241

than k23 in the trypsin-catalyzed hydrolysis. (1955).

Another possibility should be considered, however. The phe- 13. ROVERY, M., POILROUX, M., CURNIER, A., AND DESNUELLE,

nomenon of enzyme specificity would suggest that Tos-Arg-OMe, P., Biochim. Biophys. Acta, 16, 590 (1955).

14. WILCOX, P. E., KRAUT, J., WADE, R. D., AND NEURATH, H., which is a specific substrate of trypsin, binds mainly in an unpro- Biochim. Biophys. Acta, 24, 72 (1957).

ductive mode to chymotrypsin. If unproductive binding does 15. SCHONBAUM, G. It., ZERNER, B., AND BENDER, M. L., J. Biol.

predominate and interferes with nroductive binding-a situation Chem., 236, 2930 (1961).

that has been suggested as existing in other enzyme-catalyzed 16. DIXON, G. H.. AND NEURATH, H., J. BioZ. Chem.. 226. 1049

(1957). I I

reactions (35, 36)-then the condition of k23 > k34, which is ob- 17. BERNHARD, S. A., LEE, B. F., AND TASHJIAN, Z. H., J. Mol.

served in the catalytic reactions of specific substrate esters, could Biol., 18, 405 (1966).

still pertain to the Tos-Arg-OMe hydrolysis as well. Consider 18. APPLEWHITE, T., WAITE, H., AND NIEMANN, C., J. Amer.

the following mechanism : Chem. Sot.. 60. 1465 (1958).

19. HAMMES, G. b., AND H~~LA&, J. L., Biochem., 7,1519 (1968). 20. LONG, C. (Editor), Biochemists’ handbook, D. van Nostrand,

PI Princeton, New Jersey, 1961, pp. 32-36. KU Eu \ Eo + So G 7-

</ES-EPzF 21. LINEWEAVER, H., AND BURK, D., J. Amer. Chem. Sot., 66, 658

k23 k E + Pz (4)

34 (1934).

22. WILKINSON, G. N., Biochem. J., 80, 324 (1961).

where E, stands for a complex formed by unproductive binding. 23. CUNNINGHAM, L. W., AND BROWN, C. S., J. Biol. Chem., 221,

287 (1956). When steady state is reached, and with K’, > K,, the rate equa- 24. HAMMOND, B. R., AND GUTFREUND, H., Biochem. J., 61, 187

tion becomes: (1955). 25. BENDER, M. L., K~DY, F. J., AND GUNTER, C. R., J. Amer.

Chem. Sot., 86, 3714 (1964).

(5) 26. LENARD, J., JOHNSON, S. L., HYMAN, R. W., AND HESS, G. P.,

Anal. Biochem. 11, 30 (1965). 27. FOSTER, R. J., AND NIEMANN, C., J. Amer. Chem. Sot., 77.1886

I f k34 (K’,/K,) > k23, the catalytic rate constant keat = kt3 (1955).

28. TROWBRIDGE, C., KREHBIEL, A., AND LASKOWSKI, M., Bio- (Ku/K’s), and K&pp) = K,. Under these conditions, the chemistry, 2, 843 (1963).

formation of a steady state intermediate would not be observable 29. MATTHEWS, B. W., SIGLER, P. B., HENDERSON, R., AND BLOW,

in the experiments described here. D. M., Nature, 214,652 (1967).

The data obtained with Tos-Arg-OMe indicate that K,(app) 30. SIGLER, P. B., BLOW, D. M., MATTHEWS, B. W., AND HENDER-

has about the same value as the independently determined en- SON, R., J. Mol. Biol., 35, 143 (1968).

31. BLOW, D. M., BIRKTOFT, J. J., AND HARTLEY, B. S., Nature, zyme-substrate dissociation constant (see Tables III and IV). 221, 337 (1969).

Either of the conditions outlined above-k31 > k23 in the mecha- 32. HARTLEY, B. S., Nature, 201, 1284 (1964).

nism shown in Equation 1, or k23 > ks4 but with k34(K’a/Ku) > 33. MELOUN, B., KLUH, I., KOSTKOZ, V., MO~~VECK, L., PRUSIK,

kz3 in the mechanism shown in Equation 4-would lead to this Z., VANXEK, J., KEIL, B., AND S~RM, F., Biochim. Biophys. Acta, 130, 543 (1969).

observation. In the case of the first alternative, K,(app) would 34. INAGAMI, T., AND STURTEVANT, J. M., J. BioZ. Chem., 236, 1019

be a measure of K’,, a situation previously observed by us (4, 5) (1960).

in hydrolyses of chymotrypsin-specific amide substrates. In 35. BLAKE, C. C. F., JOHNSON, L. N., MAIR, G. A., NORTH,

the second case, K,(app) would be a measure of K, (Equation A. C. T., PHILLIPS, D. C., AND SARMA, V. R., Proc. Roy. SOC.

_ _ Ser. B BioZ. Sci., 167,378 (1967). 4), the dissociation constant of the unproductive enzyme com- 36. RUPLEY, J. A., Proc. Roy. Sot. Ser. B BioZ. Sci., 167,416 (1967).

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James McConn, Edmond Ku, Albert Himoe, Karl G. Brandt and George P. HessSPECIFIC SUBSTRATE ESTERS BY STOPPED FLOW TECHNIQUES

DETERMINATION OF PRE-STEADY STATE KINETIC PARAMETERS FOR Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates: V.

1971, 246:2918-2925.J. Biol. Chem. 

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