Keiichi Kihara

The thermolysin-catalyzed condensation reactions of n-substituted aspartic and glutamic acids with phenylalanine alkyl esters

Abstract The title reactions occur with concurrent peptide synthesis and optical resolutions of racemic substrates. With this method, Z-L-Asp-L-Phe-OME, the precursor to the synthetic sweetner, could be obtained in high yield.

On the mechanism of the action of thermolysin: kinetic study of the thermolysin-catalysed condensation reaction of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester

The mechanism of the thermolysin-catalysed condensation reaction of N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) with L-phenylalanine methyl ester (L-Phe-OMe) giving N-benzyloxycarbonyl-L-aspartyl-L-phenyl-alanine methyl ester, the precursor of the synthetic sweetener L-aspartyl-L-phenylalanine methyl ester (aspartame), was investigated by kinetic measurements. It was found that the reaction is first order in L-Phe-OMe and the Lineweaver–Burk plot of 1/v against 1/[Z-L-Asp] yields a straight line, showing that the reaction involves consecutive reactions of Z-L-Asp and L-Phe-OMe with the enzyme and with the Z-L-Asp–enzyme complex, with the second reaction being the rate-determining step. This fact suggests that an amino-enzyme intermediate is probably not involved in the reaction. It was also found that L-Phe-OMe neither inhibits nor enhances the reaction, whereas Z-L-Asp acts as a competitive inhibitor, and moreover the binding constants of Z-L-Asp and Z-D-Asp with the enzyme are close to each other. From these facts and other evidence, a mechanism which is the reverse of general base catalysed hydrolysis of a peptide bond is proposed for the reaction.