Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates: V. DETERMINATION OF PRE-STEADY STATE KINETIC PARAMETERS FOR SPECIFIC SUBSTRATE ESTERS BY STOPPED FLOW TECHNIQUES

Abstract

Abstract It has been suggested that the chymotrypsin-catalyzed hydrolysis of specific substrates is described by a three-step mechanism: [see PDF for equation] and that for esters k23 is much greater than k34, and K's is greater than the steady state kinetic parameter Km(app). Steady state kinetic investigations do not allow one to determine 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 chymotrypsin-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's, k23, and k34 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 presteady 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 k23, a study that is not possible with esters of aromatic acids, since in these reactions k23 becomes too large above pH 6 to be adequately measured by the stopped flow technique. The pH dependence of k23 was found to be similar to the pH dependence of k34. This information has not been available previously. p-Tosyl-l-arginine methyl ester and N-α-p-tosyl-l-argininamide 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. Unlike in the chymotrypsin-catalyzed hydrolysis of specific substrate esters, the accumulation 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 k34 is rate-limiting. It is shown that unproductive binding of the unspecific substrate can also account for the data.