The role of the medium in solvent isotope effects on serine protease action

Abstract

The hydrolysis of N-carbobenzyloxyaminoacyl-O-p-nitrophenyl esters derived from L-leucine, L-phenylalanine, and L-tryptophan, with catalysis by bovine pancreatic α-chymotrypsin at pH 7.00 at 25.00°C in water containing acetonitrile from 15.0% to 60% (v/v), exhibits values of kcat/Km that decrease as the 34th to 36th power of the activity of water and values of kcat that are essentially constant. Solvent isotope effects kcat(H2O)/ kcat(D2O) range from 2.1 to 3.0 with proton inventories (dependences of kcat(n) on n, the atom fraction of deuterium in the water component of the solvent) that are linear, regardless of the amount of acetonitrile in the medium. The isotope effect on kcat thus appears to arise from a single transition-state site and to be little affected by the medium. Contributions from the medium to solvent isotope effects on enzymic reactions clearly exist but appear not to be detectable in the deacylation reactions of acyl chymotrypsins.Key words: solvent isotope effects, proton inventories, medium effects, enzyme catalysis, alpha-chymotrypsin.