Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid.

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme-catalysed conversion of D-Tyr-Val-Gly to D-Tyr-Val-NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the alpha-H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non-peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates. The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide-amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co-chromatography in three chromatographic systems. The results demonstrate that the enzyme-catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.