Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates

Pre-steady state kinetic parameters pertaining to chymotrypsin-catalyzed hydrolysis of specific substrate esters can be obtained from measurements of the displacement of the competitive inhibitor proflavin from enzyme by substrate. This method, which was previously used in experiments with specific substrate amides, has now been applied to the investigation of N-acetyl-l-phenylalanine methyl ester, N-acetyl-l-tryptophan ethyl ester, and N-furylacryloyl-l-tyrosine ethyl ester. Results were found to be in agreement with data obtained by established procedures including measurement of spectral changes of the enzyme upon formation of complexes with substrate and direct measurement of product formation. All available data on chymotrypsin-catalyzed hydrolysis of specific substrate esters are consistent with the previously proposed mechanism [see PDF for equation] in which P1 represents an alcohol and P2 an acid.