Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of PHM was found not to extend beyond Asp359. Shorter PHM proteins were eliminated intracellularly, suggesting that they failed to fold correctly. A set of mutant PHM proteins whose design was based on the structural and mechanistic similarities of PHM and dopamine beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/D beta M homology domain eliminated activity, while deletion of a third His-rich cluster unique to PHM failed to affect activity; the catalytically inactive mutant PHM proteins still bound to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized PHM indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of PHM and D beta M. Mutation of the Met residues common to PHM and D beta M to Ile identified Met314 as critical for catalytic activity.

Peptidylglycine ␣-amidating monooxygenase (PAM) catalyzes the carboxyl-terminal amidation of bioactive peptides through a two-step reaction involving the monooxygenase and lyase domains. PAM undergoes endoproteolytic cleavage in neuroendocrine cells in the lyase domain. To determine which of the two possible paired basic sites is utilized, truncated PAM proteins ending at these sites were stably expressed in Chinese hamster ovary cells. While characterizing the truncation mutants, it became apparent that N-glycosylation occurred post-translationally at the single site localized near the carboxyl terminus of the lyase domain. The post-translational N-glycosylation of this site does not require the newly synthesized protein to remain tightly bound to membranes. Both malfolded, secretion incompetent proteins and fully active, secreted proteins were subject to post-translational N-glycosylation.