investigations of the chymotrypsin-catalyzed hydrolysis of ... ?· hypothesis that the ph...
Post on 10-Mar-2019
Embed Size (px)
THE JOURNAL OF BIOLOGICAL CHEM~~Y Vol. 242, No. 5, Issue of March 10, PP. 919-929, 1967
P&ted in U.S.A.
Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates
I. THE pH DEPENDENCE OF THE CATALYTIC HYDROLYSIS OF N-ACETYL-L-TRYPTOPHANAMIDE BY THREE FORMS OF THE ENZYME AT ALKALINE pH*
(Received for publication, July 7, 1966)
ALBERT HIMOE,~ PAUL C. PARKS,~ AND GEORGE P. HESS~
From the Xection of Biochemistry and Molecular Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14850
The pH dependence of the steady state kinetic parameters K,(app) and $,t of the a-, acetylated 6-, and ar-chymotrypsin- catalyzed hydrolysis of the specific amide substrate N-acetyl- r.-tryptophanamide, has been investigated in the neutral and alkaline pH regions. A completely automatic technique for measuring the hydrolysis of amides was used. New results and important aspects of the chymotrypsin-catalyzed hy- drolysis of a specific amide substrate which have emerged from this investigation are the following.
The pH dependence of the catalytic reaction at alkaline pH is adequately accounted for by the pH dependence of K,(app). In earlier experiments we have shown that in amide hydrolysis the steady state kinetic parameter K,(app) is a measure of an over-all enzyme-substrate dissociation constant (K,). (a) K,(app) was observed to be pH-de- pendent in the hydrolysis of N-acetyl-r&yptophanamide as catalyzed by all three forms of the enzyme. Analysis of this pH dependence shows that an ionizing group with pK(app) - 8.5 is involved; an ionizing group of the enzyme with this pK(app) has been implicated in all chymotrypsin-catalyzed reactions that have been studied. (b) The catalytic rate constant kat was found to be pa-independent in the pH region 8 to 10 for the &chymotrypsin-catalyzed reaction and in the pH region 8 to 9.2 for the a-chymotrypsin-catalyzed reaction. (c) The pH dependence of the steady state kinetic parameters kcat and K,(app) in the chymotrypsin-catalyzed hydrolysis of iV-acetyl-L-tryptophanamide is consistent with the findings of our earlier studies of the individual steps in the reaction between chymotrypsin and diisopropyl fluoro- phosphate and in the chymotrypsin-catalyzed hydrolysis of N-acetyl-L-phenylalaninamide: studies which indicated that the pH dependence of chymotrypsin-catalyzed hydrolysis above pH 8 is due to the effect of pH on the formation of
* We are grateful to the National Institutes of Health and the National Science Foundation for financial support.
1 United States Public Health Service Postdoctoral Trainee. 5 National Institutes of Health Postdoctoral Fellow. f[ To whom reprint requests should be addressed at 210 Savage
chymotrypsin complexes and not on the bond-breaking step. (d) The experiments reported here are not in agreement with earlier studies of Bender and his co-workers, which indicated that the pH dependence of chymotrypsin-catalyzed reactions above pH 8 is due to the effect of pH on the bond- breaking step, the formation of chymotrypsin-substrate complexes being pa-independent.
The 6 form of chymotrypsin is a more efficient catalyst than the (Y form in the hydrolysis of N-acetyl-L-tryptophanamide, as indicated by both the observed maximum rate of reaction and the binding of substrate to enzyme. Both the values and the pH dependencies of the steady state kinetic parameters are different for these two forms of the enzyme, but are the same for 6- and acetylated b-chymotrypsin. Only limited and qualified information regarding differences in the catalytic properties of a- and d-chymotrypsin has been previously available.
These and previous experiments are consistent with the hypothesis that the pH dependence of chymotrypsin-cata- lyzed reactions at alkaline pH is due to a pH-dependent equilibrium between two major conformations of the enzyme, with the same ionizing group of pK (app) - 8.5 controlling both the equilibrium between enzyme conformations and the decrease in catalytic properties of the enzyme. This hypothe- sis leads to several predictions, discussed in this paper, which can be tested experimentally.
Only limited and qualified information regarding chymo- trypsin-catalyzed reactions at alkaline pH is available (1). It is known that the catalytic properties of the enzyme toward both specific and model substrates decrease above pH 8 (2-4). Steady state kinetic experiments with cY-chymotrypsin and specific substrate esters and amides have indicated that an ionizing group of the enzyme with pK(app) - 8.5 is implicated in this decrease of enzymic activity (5, 6). Recent steady state kinetic experiments have led to the conclusion (5-9) that the pH de- pendence of chymotrypsin-catalyzed reactions at alkaline pH values is due to the effect of hydrogen ion concentration on the
by guest on March 9, 2019
920 Chymotrypsin-catalyzed Hydrolysis of Specific Substrates. 1 Vol. 242, No. 5
bond-breaking step and that the formation of enzyme-substrate complexes is pH-independent. However, this conclusion ap- peared questionable in view of investigations (10-22) of the individual steps in the stoichiometric reaction of chymotrypsin with its specific inhibitor DFPl and in the chymotrypsin-cata- Iyzed hydrolysis of N-acetyl-L-phenylalaninamide. It has been shown that the chymotrypsin-DFP reaction is similar to chymo- trypsin-catalyzed hydrolysis of specific substrates in that its reaction rate also decreases above pH 8 (10, 20, 23), with implica- tion in this decrease of an ionizing group of the enzyme of
ok - 9 (20). Kinetic studies of individual steps in the DFP reaction (19, 20), made through measurements of phos- phorylation rate and changes in the spectral and optical rotatory properties of the enzyme (10-19) suggested, however, a different mechanism for the pH dependence above pH 8 than the one proposed (5-9) for substrate hydrolysis. The DFP studies indicated that the observed decrease in phosphorylation rate above pH 8 is due to the effect of pH on a chymotrypsin-inhibitor complex which is formed by a reversible process which precedes the phosphorylation step. A similar effect of pH on enzyme- substrate complex formation was deduced from studies of in- dividual steps in the chymotrypsin-catalyzed hydrolysis of N-acetyl-n-phenylalaninamide (22). The nature of this pH effect has been suggested by experiments (17, 21) with acetylated &chymotrypsin which suggest that the level of pH affects an equilibrium between active and inactive conformations of the enzyme, and thereby the formation of enzyme complexes and the catalytic properties of the enzyme.
Because of these experimental findings, we are now conducting an investigation of chymotrypsin-catalyzed reactions of specific substrates at alkaline pH values. As a first step in these studies, we have measured the pH dependence of the steady state kinetic parameters, K,(app) and kcat, of the 6- and acetylated d-chymo- trypsin-catalyzed hydrolysis of the specific amide substrate ATA, and have reinvestigated the previously studied (6) (Y- chymotrypsin-catalyzed hydrolysis of the same substrate. Acetylated &chymotrypsin was used in this work because of previous evidence (21) that this enzyme exhibits, above pH 8, a pH-dependent equilibrium between two major conformations; the c+ and &chymotrypsin were used for purposes of comparison. An amide was chosen as specific substrate because recent experi- ments (24) indicated that in the chymotrypsin-catalyzed hy- drolysis of amides, but not esters, K,(app) is a direct measure of the enzyme-substrate equilibrium constants. Observation of a pH dependence of K,(app) in the hydrolysis of ATA would therefore indicate a pH dependence in the formation of enzyme- substrate complexes.
The measurements reported in this study were greatly facili- tated by the development of a completely automatic technique for measuring the hydrolysis of amides (25), a technique which as far as enzyme concentration, precision of measurement, and time involved are concerned, compares favorably with the pa-stat method used for measuring the kinetics of ester hydrolysis. Steady state kinetic parameters of 6- or acetylated b-chymo- trypsin-catalyzed hydrolysis of a specific amide substrate have not been reported prior to this study. A preliminary report of a part of this investigation has appeared (26).
1 The abbreviations used are: DFP, diisopropyl fluorophos- phate; ATA, N-acetyl-n-tryptophanamide.
Three times crystallized chymotrypsinogen (Lots 703, 781, and 8936) and three times crystallized, salt-free cr-chymotrypsin were obtained from Worthington. The &chymotrypsin was prepared just prior to each kinetic run by activating chymo- trypsinogen under conditions that are known to lead essentially to the 6 form of the enzyme (27). The ar-chymotrypsin and some of the &chymotrypsin preparations were passed through Sephadex columns before use, as suggested by Yapel et al. (28). The procedure was to dissolve 100 mg of enzyme in approximately 10 ml of cold 1 mM HCl solution, pass the solution through a Sephadex (G-25 coarse) column (90 X 2.5 cm) which had been equilibrated with 1 mu HCl, elute the sample with 1 mM HCl in a cold room (-4), and collect the first fraction emerging from the column for use in the experiments. No difference in enzyme properties was noted between b-chymotrypsin samples which had been passed through Sephadex columns and those which had not.
Acetylated &chymotrypsin was prepared by acetylating chymotrypsinogen and then activating it, as describ