Identification of two active site residues in human angiotensin I-converting enzyme.
Angiotensin I-converting enzyme (ACE) contains two zinc-dependent catalytic domains (N and C domains) each bearing the motif HEXXH where the two histidines form two of the three amino acid zinc ligands. Sequence alignment of each ACE domain with other zinc metalloproteases, indicates a glutamate residues which putatively constitutes the third zinc ligand and an aspartate residue which may form an indirect zinc interaction. We investigated the functional roles of the glutamate and aspartate residues in the ACE C domain (Glu987 and Asp991) using a cDNA encoding an inactive N domain. We mutated Glu987 to aspartate (E987D) or valine (E987V) and Asp991 to glutamate (D991E) or alanine (D991A). Catalytically active mutants (E987D, D991E and D991A) exhibited similar Km values for hippuryl-His-Leu compared to non-mutated C domain. E987D displayed a 300-fold decrease in kcat and a 25-fold reduction in sensitivity to the ACE inhibitor trandolaprilat, whose binding is zinc-dependent. E987V was catalytically inactive and did not bind [3H]trandolaprilat. D991E and D991A exhibited a 3.8- and 22-fold decrease in kcat, respectively, and the Ki' values for trandolaprilat were increased 8- and 29-fold. These results provide strong evidence that Glu987 constitutes the third zinc ligand in the ACE C domain and suggest a role for Asp991 in positioning the C domain active site zinc ion.
INSERM Unité 36, Collège de France, Paris.
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D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D018076:DNA, Complementary; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D000806:Angiotensin-Converting Enzyme Inhibitors; D018076:DNA, Complementary; D001224:Aspartic Acid; D018698:Glutamic Acid; D007703:Peptidyl-Dipeptidase A