Kinetics of trypsin catalysis in the industrial conversion of porcine insulin to human insulin.
The kinetics of coupling and transpeptidation reactions catalysed by trypsin were studied in media with low water contents to see whether the usual Michaelis-Menten kinetics applied under the unusual conditions used in peptide bond synthesis, to obtain information about the magnitude of rate constants and activation energies, and to gain insight into the mechanism of catalysis. Porcine insulin, des-(AlaB30)-porcine insulin, human insulin-ThrB30-OMe and human insulin-ThrB30(But)-OBut were used as substrates. Two threonine esters (Thr-OMe and Thr(But)-OBut) were used for transpeptidation and coupling. The reactions progressed according to first-order kinetics until about 35% conversion, and the experimental data were adequately explained by Michaelis-Menten kinetics. The rates of the coupling and transpeptidation reactions in media with low water contents were orders of magnitude below the rates of peptide bond hydrolysis by trypsin in water. It was not possible to approach saturation of the enzyme with substrate so determination of Km was impossible, but for each substrate a value larger than 0.1 M was estimated from the Lineweaver-Burk plot. The rate of release of alanine from porcine insulin depended on the type of threonine ester present; for example, Thr-OMe inhibited the reaction. Coupling occurred faster than transpeptidation. However, in the medium used, the activation energies for the two reactions were similar (about 50 kJ/mol), so the difference in reaction rates is probably due to different transmission coefficients in the activated transition states. Computer simulations enabled us to obtain quantitative descriptions of the reaction progress curves from fitted rate constants.
D000409:Alanine / Q000032:analysis; D002384:Catalysis; D003201:Computers; D006801:Humans; D007328:Insulin / Q000138:chemical synthesis*; D061389:Insulin, Regular, Pork; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014357:Trypsin / Q000378:metabolism*
D007328:Insulin; D061389:Insulin, Regular, Pork; C000006:insulin, neutral; D014357:Trypsin; D000409:Alanine