Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of these diverse physiological regulators. The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states. The anomalous signal of the endogenous coppers was used to determine the structure of the catalytic core of oxidized rat PHM with and without bound peptide substrate. These structures strongly suggest that the PHM reaction proceeds via activation of substrate by a copper-bound oxygen species. The mechanistic and structural insight gained from the PHM structures can be directly extended to dopamine beta-monooxygenase.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.

In vertebrates, the two-step peptide alpha-amidation reaction is catalyzed sequentially by two enzymatic activities contained within one bifunctional enzyme called PAM (peptidylglycine alpha-amidating mono-oxygenase). Drosophila head extracts contained both of these PAM-related enzyme activities: a mono-oxygenase (PHM) and a lyase (PAL). However, no bifunctional PAM protein was detected. We identified cDNAs encoding an active mono-oxygenase that is highly homologous to mammalian PHM. PHM-like immunoreactivity was found within diverse larval tissues, including the CNS, endocrine glands, and gut epithelium. Northern and Western blot analyses demonstrate RNA and protein species corresponding to the cloned PHM, but not to a bifunctional PAM, leading us to predict the existence of separate PHM and PAL genes in Drosophila. The Drosophila PHM gene displays an organization of exons that is highly similar to the PHM-encoding portion of the rat PAM gene. Genetic analysis was consistent with the prediction of separate PHM and PAL gene functions in Drosophila: a P element insertion line containing a transposon within the PHM transcription unit displayed strikingly lower PHM enzyme levels, whereas PAL levels were increased slightly. The lethal phenotype displayed by the dPHM P element insertion indicates a widespread essential function. Reversion analysis indicated that the lethality associated with the insertion chromosome likely is attributable to the P element insertion. These combined data indicate a fundamental evolutionary divergence in the genes coding for critical neurotransmitter biosynthetic enzymes: in Drosophila, the two enzyme activities of PAM are encoded by separate genes.