PEPTIDYLGLYCINE ALPHA-HYDROXYLATING MONOOXYGENASE - ACTIVE-SITE RESIDUES, DISULFIDE LINKAGES, AND A 2-DOMAIN MODEL OF THE CATALYTIC CORE

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, a scorbate, and molecular oxygen dependent enzyme that catalyzes the fir st step leading to the C-terminal amidation of glycine-extended peptid es. The catalytic core of PHM (PHMcc), refined to residues 42-356 of t he PHM protein, was expressed at high levels in CHO (DG44) (dhfr(-)) c ells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing cond itions cleaved the protein at Lys(219), indicating that the protein co nsists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-li nked peptides generated by sequential CNBr and pepsin treatment of rad iolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal d omain (Cys(47)-Cys(186), Cys(81)-Cys(126), and Cys(114)-Cys(131)), alo ng with three of the His residues critical to catalytic activity (His( 107), His(108), and His(172)). Two disulfide linkages (Cys(227)-Cys(33 4), 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 t he 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 d ecreased the the k(cat) 7-fold, again identifying this region of the C -terminal domain as critical to catalytic activity. Taking into accoun t information on the copper ligands in PHM, we propose a two-domain mo del with a copper site in each domain that allows spatial proximity be tween previously described copper ligands and residues identified as c atalytically important.