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) is a mono-zinc-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 used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic 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 serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.
Cottrell
G S
GS
Proteolysis Research Group, School of Biochemistry and Molecular Biology, and Astbury Centre for Structural Molecular Biology, The University of Leeds, Leeds LS2 9JT, United Kingdom.
Hyde
R J
RJ
Lim
J
J
Parsons
M R
MR
Hooper
N M
NM
Turner
A J
AJ
D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000235:genetics; D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D020134:Catalytic Domain; D003198:Computer Simulation; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000600:Amino Acids, Dicarboxylic; D008024:Ligands; D008565:Membrane Proteins; D009842:Oligopeptides; D006639:Histidine; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D001920:Bradykinin