Peptidylglycine alpha-hydroxylating monooxygenase: Active site residues, disulfide linkages, and a two-domain model of the catalytic core

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 Lys(219), 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 (Cys(47)-Cys(186), Cys(81)-Cys(126), and Cys(114)-Cys(131)), along with three of the His residues critical to catalytic activity (His(107), His(108), and His(172)). Two disulfide linkages (Cys(227)-Cys(334), and Cys(293)-Cys(315)) occur in the C-terminal domain, along with the remaining two essential His residues (His(242), His(244)) and Met(314), thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr(79) or Tyr(318) with Phe increased the K-m of PHM for its peptidylglycine substrate without affecting the V-max. Replacement of Glu(313) with Asp increased the K-m 8-fold and decreased the the k(cat) 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.