Recombinant type A rat 75-kDa α-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an α-hydroxyglycine intermediate

Abstract

The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine α-amidating enzyme (α-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A α-AE secrete the expected 75-kDa enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-TyrValGly and dansyl-TyrValα-hydroxyglycine to dansyl-TyrValNH 2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A α-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-α-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second enzyme to convert peptidyl-α-hydroxyglycine to peptidyl-NH 2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.