Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates.

摘要: The effects of pressure on the trypsin-catalyzed hydrolyzed hydrolyses three specific substrates, N-benzoyl-L-arginine ethyl ester (BzArgOEt), amide (BzArgNH2) and p-nitroanilide (BzArgNA), have been examined. volume activation (delta V++) for kcat was -2.4 ml/mol BzArgOEt +3 - +6 BzArgNH2. Because different rate-determining steps in steady-state kinetics, delta V++ value would indicate deacylation step, whereas that BzArgNH2 acylation step. volumes were accounted terms difference mechanisms formation decomposition tetrahedral-like intermediates during steps. V values BzArgNH2- thionine-trypsin complexes several ml/mol, consistent with fact main driving force substrate binding to this enzyme is electrostatic interaction, contrast alpha-chymotrypsin complex indole (approximately 0 ml/mol) or 2-furylacryloyl-D-tryptophan methyl ml/mol), which hydrophobic interaction dominant binding. For hydrolysis BzArgNA, showed a distinct at high concentrations, dependence four parameters, ks, Ks, (the catalytic rate dissociation constant normal enzyme-substrate complex, respectively), Kss (those activated by second molecule), measured 1 atm 1000 (25 degrees C). All parameters increased increase pressure.