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THIJOUBNAL ~~BIOLO~~ALCREF~~ISTRY Vol.242, No.17,Iasue of &ptembezlO,pp. 397%3992,1967
Printed in U.S.A.
Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates
III. DETERMINATION OF INDIVIDUAL RATE CONSTANTS AND ENZYME-SUBSTRATE BINDING CONSTANTS FOR SPECIFIC AMIDE AND ESTER SUBSTRATES*
(Received for publication, February 23, 1967)
KARL G. BRANDT,$ ALBERT HIMOE,~ AND GEORGE P. HESS~
From the Section of Biochemistry and Molecular Biology, Division of Biological Sciences, Cornell University. Ithaca, New York: 14.860
Enzyme-substrate binding constants, K’e, for chymotrypsin and specific amide (N-acetyl-Mryptophanamide and N- acetyl-L-phenylalaninamide) and ester (N-acetyl-L-tryp- tophan ethyl ester) substrates have been measured by a proflavin-displacement method. Also, the rate constant for the formation of one intermediate in the chymotrypsin- catalyzed hydrolysis of the ester has been determined at selected pH values. These constants have not previously been determined.
The proflavin-displacement method used to measure the enzyme complex concentration has previously been found to yield results in agreement with those obtained by measuring the spectral changes of the enzyme at 290 mp. These spectral changes at 290 rnp, observed in all chymotrypsin- catalyzed reactions studied, have been shown to arise during the reversible formation of an enzyme-substrate complex which precedes the bond-breaking step.
New information important to an understanding of chymo- trypsin-catalyzed hydrolysis in general has been obtained from this work. It is shown directly, for the Crst time, that in the chymotrypsin-catalyzed hydrolysis of at least two specific amide substrates, the rate-limiting bond-breaking step follows the reversible formation of an enzyme-substrate complex with a dissociation constant K’s, and that Kls and the steady state kinetic parameter K, (app) are equivalent. The pH-dependent rate constant (ks) measured in the chymotrypsin-catalyzed hydrolysis of the ester we have studied is shown to pertain to a second intermediate detected
* This research was supported by grants from the National Institutes of Health and the National Science Foundation.
$ National Institutes of Health Postdoctoral Fellow. Present address, Department of Biochemistry, Purdue University, Lafayette, Indiana 47907.
6 United States Public Health Service Postdoctoral Trainee. Present address, Department of Biochemistry, Baylor University College of Medicine. Houston. Texas 77025.
7 To whom reprint reques& should be addressed at 210 Savage Hall, Cornell University, Ithaca, New York 14850.
in this reaction, an intermediate not seen in the chymotrypsin- catalyzed hydrolysis of the specific substrate amides.
The K’, values at pH 8.0 for the complexes of cu-chymo- trypsin with N-acetyl-Mryptophanamide and N-acetyl-L- phenylalaninamide were found to be 4.7 nmr and 29 mu, respectively, in excellent agreement with the steady state kinetic parameter K, (app). In the case of the cr-chymo- trypsin-catalyzed hydrolysis of N-acetyl-Mryptophan ethyl ester, data on the pH dependence of k23 was found to be consistent with a pK (app) value of 6.6 for the group of the enzyme controlling the rate, and with a limiting pH-inde- pendent value for k23 of 1800 see-? The K/e value for this ester and a-chymotrypsin at, for instance, pH 5.0, was found to be 2.3 m&r, while K, (app) has been reported as -0.08 mM. Values of K’, and k28, determined in these experiments, together with previously determined values of the steady
. . state kmetm parameter, &, allowed a calculation of K,,, (app). At pH 5.0, this calculated value is -0.06 mu, in reasonable agreement with the experimentally observed value.
Investigations of chymotrypsin-catalyzed reactions, although extensive, have relied almost exclusively on the steady state kinetic approach, and therefore very little is known about the individual steps in these reactions (1). We have undertaken an investigation of these steps in order to gain further understanding of the catalytic processes involved in the reactions.
The work was begun with a study of the reaction of chymo- trypsin with its specific inhibitor diisopropyl fluorophosphate and with model substrates &lo), because these reactions, in contrast to chymotrypsin-catalyzed hydrolyses of specific substrates, are known to proceed via long lived, covalently bonded chymotrypsin compounds (11-13). Physical changes in the enzyme which were observed to accompany these reactions were characterized; these include spectral changes near 290 rncc (2, 5), changes in optical
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Chymotrypsin-catalyzed Hydrolysis of XpeciJic Substrates. III Vol. 242, No. 17
rotatory properties (4, 6, 14, 15), and changes in the acid disso- ciation constant of an ionizing group with a pK (app) of about 8.5 (4, 9, 10, 16, 17). Further information was obtained from kinetic investigations (8, 9) of the physical changes of the enzyme which accompany its stoichiometric reaction with diisopropyl fluorophosphate. We have now extended these investigations of individual reaction steps to the chymotrypsin-catalyzed hydrol- ysis of specific substrates, ester or amide derivatives of aromatic amino acids. In these reactions, only transient enzyme-sub- strate complexes are known to appear.
In an accompanying paper (18) we report preliminary kinetic as well as static equilibrium investigations of the spectral changes of the enzyme near 290 rnp which result when chymotrypsin binds the specific amide substrate N-acetyl-n-phenylalaninamide. These measurements (18) permitted the determination of an over- all enzyme-substrate dissociation constant, K’s, and the pH dependence of this constant. Extension of the experimental method used in these studies to the chymotrypsin-catalyzed hydrolysis of specific substrates which are derivatives of tyrosine or tryptophan is, however, precluded by the high absorbance of these residues in the 290 rnp region. We have therefore used another approach to the determination of K’s values and to the investigation of the pre-steady state kinetics of the chymotryp- sin-catalyzed hydrolysis of specific substrates: a spectro- photometric procedure based on the competitive binding of the dye proflavin (3,6-diaminoacridine) by the enzyme.
The competitive inhibition of chymotrypsin by proflavin, first reported by Wallace, Kurtz, and Niemann (19), has been used by Weiner and Koshland (20) and Weiner et al. (21) as the basis of a determination of dissociation constants of complexes between chymotrypsin and “virtual” substrates by equilibrium dialysis techniques. This approach cannot be extended to real substrates because these compounds become hydrolyzed during the time required for equilibrium dialysis. In the present study, a spec- trophotometric technique is used to measure, in the presence and absence of substrate, a perturbation of the absorption spectrum of proflavin which occurs upon binding to chymotrypsin. This perturbation, first reported by Bernhard and Lee (22) and Bernhard, Lee, and Tashjian (23), has been used to measure the dissociation constants for the proflavin complexes of both tryp- sin and chymotrypsin (24, 25), and for the detection of transient compounds in trypsin- and chymotrypsin-catalyzed reactions (23, 25). The magnitude of the difference spectrum, exhibiting a maximum at 465 rnp, which appears when proflavin binds to chymotrypsin, is proportional to the amount of enzyme-proflavin complex (22, 23). Introduction into the system of a substrate which competes with proflavin for the enzyme decreases the amount of enzyme-proflavin complex. Also, the substrate itself forms a weak complex with proflavin which absorbs at 465 rnp. Accordingly, the observed reduction of intensity of the difference spectrum depends on the relative concentrations of substrate and inhibitor, the dissociation constants of their complexes with the enzyme, the proflavin-substrate dissociation constant, and the molar absorption coefficients of the various proflavin complexes. Since the constants pertaining to the enzyme- and substrate- proflavin complexes can be determined independently, the chymotrypsin-substrate dissociation constant, K’,, can be calcu- lated.
In the experiments reported in this paper, dissociation con- stants were determined at selected pH levels for the complexes of chymotrypsin with the amide and the ethyl ester of N-acetyl-
n-tryptophan, and with the amides of N-acetyl-n-phenylalanine and N-acetyl-n-phenylahmine. We are also reporting the use of the proflavin-displacement method for determination of the pre- steady state kinetics in the cu-chymotrypsin-catalyzed hydrolysis of N-acetyl-n-tryptophan ethyl ester over an extended pH range. This work includes the determination of rate constants not previously accessible experimentally.
In the experiments with specific amide substrates, static deter- minations could be made, since with amides the extent of hydroly- sis during the time required for measurement is negligible. The experiments with the ester, however, required the use of stopped- flow techniques.
A preliminary report of a part of these data has appeared (26).
Materials and Apparatus
Salt-free oc-chymotrypsin crystallized three times (Lots CDI 6148-9, CD1 6127-8, CD1 6164, and CDI 6094~5), trypsin crystallized twice, and er