title: Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.
abstract: Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.
authors.affiliation: Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama, 351-0198 Japan.
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Goto
authors.forename: Akira
authors.initials: A
authors.lastname: Hattori
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Mizutani
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tsujimoto
mesh_terms: D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001794:Blood Pressure / Q000502:physiology; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002766:Cholecystokinin / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D043384:Glutamyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007609:Kallidin / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D015287:Neurokinin B / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000804:Angiotensin II; D001224:Aspartic Acid; D007609:Kallidin; D015287:Neurokinin B; D002766:Cholecystokinin; D043384:Glutamyl Aminopeptidase; D002118:Calcium