Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4.11.9) i s a monozinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was use d to align the sequence of porcine membrane-bound AP-P with other members o f the peptidase dan MG, including Escherichia coil AP-P and methionyl amino peptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoreti c blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to s erve as metal ion ligands in the active site, and mutagenesis of these resi dues resulted in fully glycosylated proteins that were catalytically inacti ve. Mutation of His429 and His532 also resulted in catalytically inactive p roteins, and these residues, by analogy with E. coli AP-P, are likely to pl ay a role in shuttling protons during catalysis. These studies indicate tha t mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase dan MG, which is compatible with ei ther a dual metal ion model or a single metal ion in the active site. The l atter model is consistent, however, with the known metal stoichiometry of b oth the membrane-bound and cytosolic forms of AP-P and with a recently prop osed model for methionyl aminopeptidase.