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  • 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).

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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 Radiomete

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