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J. Biol. Chem., Vol. 282, Issue 51, 37074-37081, December 21, 2007

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Asparatic Acid 221 Is Critical in the Calcium-induced Modulation of the Enzymatic Activity of Human Aminopeptidase A^*

Yoshikuni Goto, Akira Hattori¹, Shigehiko Mizutani, and Masafumi Tsujimoto²

From the Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama, 351-0198 Japan and the Department of Medical Science of Proteases, Nagoya University School of Medicine, Showa, Nagoya 466-8550, Japan

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.

Received for publication, August 29, 2007 , and in revised form, October 5, 2007.

^* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and a grant for the "Chemical Biology Research Program" from RIKEN. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of System Chemotherapy and Molecular Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.

² To whom correspondence should be addressed. Tel.: 81-48-467-9370; Fax: 81-48-462-4670; E-mail: tsujimot{at}riken.jp.

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