Investigation of diffusion-limited rates of chymotrypsin reactions by viscosity variation

Abstract

The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for 3 substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of kcat/Km (pH 8.0, 25.degree. C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 .times. 107 M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 .times. 105 M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 .times. 107 and 1 .times. 107 M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at .apprx. 40% and by the methyl ester at .apprx. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.