Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates.
The effects of pressure on the trypsin-catalyzed hydrolyzed hydrolyses of three specific substrates, N-benzoyl-L-arginine ethyl ester (BzArgOEt), amide (BzArgNH2) and p-nitroanilide (BzArgNA), have been examined. The volume of the activation (delta V++) for kcat was -2.4 ml/mol for BzArgOEt and +3 - +6 ml/mol for BzArgNH2. Because of different rate-determining steps in the steady-state kinetics, the delta V++ value for BzArgOEt would indicate the activation volume of the deacylation step, whereas that for BzArgNH2 the delta V++ for the acylation step. The activation volumes were accounted for in terms of the difference in the mechanisms on the formation and decomposition of the tetrahedral-like intermediates during the acylation and deacylation steps. The delta V values for the formation of BzArgNH2- and thionine-trypsin complexes were several ml/mol, consistent with the fact that the main driving force of the substrate binding to this enzyme is electrostatic interaction, and in contrast to the delta V values of alpha-chymotrypsin complex formation with indole (approximately 0 ml/mol) or 2-furylacryloyl-D-tryptophan methyl ester (approximately 0 ml/mol), for which the hydrophobic interaction is the dominant force of the substrate binding. For the hydrolysis of BzArgNA, which showed a distinct substrate activation at high substrate concentrations, the pressure dependence of the four parameters, ks, Ks, (the catalytic rate and dissociation constant of the normal enzyme-substrate complex, respectively), Kss and Kss (those of the complex activated by the binding of the second substrate molecule), were measured at 1 atm and 1000 atm (25 degrees C). All of the four parameters increased with increase in pressure.
D000577:Amides; D000814:Aniline Compounds; D000818:Animals; D001120:Arginine / Q000378:metabolism / Q000031:analogs & derivatives; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006874:Hydrostatic Pressure; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism*
D000577:Amides; D000814:Aniline Compounds; C008625:nitroaniline; D001120:Arginine; C038028:N(alpha)-benzoylarginineamide; C012208:benzoyl L-arginine methyl ester; C009727:benzoylarginine ethyl ester; D014357:Trypsin