Dec 25, 1971
The Journal of biological chemistry
Abstract The α-chymotrypsin-catalyzed hydrolysis of the p-nitroanilides of glutaryl- and succinyl-l-phenylalanine and of N-acetyl-l-tyrosine has been studied over a range of enzyme concentrations in which the percentage of monomer decreases from more than 99.8% to less than 34%. The measurements were made at 22.5° and pH 6.2 in 0.2 m ionic strength phosphate buffer, so that the results of an ultracentrifuge study of α-chymotrypsin oligomerization (Rao, M. S. N., and Kegeles, G., J. Amer. Chem. Soc., 80, 5724 (1958)) could be used to calculate the distribution of the enzyme among monomeric, dimeric, and trimeric forms. The hydrolysis of all three substrates was found to be first order in substrate concentration and first order in monomer concentration. Within experimental error, second order rate constants deduced from stopped flow measurements at high enzyme concentrations by assuming that oligomers are inactive were found to equal those derived from the effect of substrate concentration on catalysis by monomeric α-chymotrypsin. The simplest interpretation of the results of this study is that only monomeric α-chymotrypsin is catalytically active. This conclusion contradicts an earlier report (Inagami, T., and Sturtevant, J. M., Biochemistry, 4, 1330 (1965)) that oligomers must be assumed active in the α-chymotrypsin-catalyzed hydrolysis of N-acetyl-l-tyrosine p-nitroanilide.