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

The hydrolysis of N-carbobenzyloxyaminoacyl-O-p-nitrophenyl esters derived from L-leucine, L-phenylalanine, and L-tryptophan, with catalysis by bovine pancreatic a-chymotrypsin at pH 7.00 at 25.00 degrees C in water containin g acetonitrile from 15.0% to 60% (v/v), exhibits values of k(cat)/K-m that decrease as the 34th to 36th power of the activity of water and values of k (cat) that are essentially constant. Solvent isotope effects k(cat)/(H2O)/ k(cat)(D2O) range from 2.1 to 3.0 with proton inventories (dependences of k (cat)(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 k(cat) thus appears to arise from a single tr ansition-state site and to be little affected by the medium. Contributions from the medium to solvent isotope effects on enzymic reactions clearly exi st but appear not to be detectable in the deacylation reactions of acyl chy motrypsins.