The reaction product of peptidylglycine alpha-amidating enzyme is a hydroxyl derivative at alpha-carbon of the carboxyl-terminal glycine

The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide. We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography. With the purified enzyme, we detected an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis. The production of the intermediate required copper, oxygen, and ascorbate and increased linearly with incubation time. The structure of the intermediate was determined to be a hydroxyl derivative at the carboxyl-terminal glycine by fast atom bombardment mass spectrometry and by proton NMR. The intermediate was readily converted into an alpha-amidated product in alkaline conditions in a nonenzymic fashion. The nonenzymic conversion required no cofactor but was extremely accelerated by the addition of copper ion or at higher temperature. Our data suggest that the direct product of the alpha-amidating reaction is not an alpha-amidated peptide but a hydroxyl derivative at the alpha-carbon of the carboxyl-terminal glycine.