ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.
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Research Article|
October 28 2008
Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1
Yoshikuni Goto;
Yoshikuni Goto
*Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
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Hiroe Tanji;
Hiroe Tanji
*Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
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Akira Hattori;
Akira Hattori
†Department of System Chemotherapy and Molecular Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, 606-8501, Japan
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Masafumi Tsujimoto
Masafumi Tsujimoto
1
*Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
1To whom correspondence should be addressed (email tsujimot@riken.jp).
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Publisher: Portland Press Ltd
Received:
May 15 2008
Revision Received:
June 25 2008
Accepted:
July 01 2008
Accepted Manuscript online:
July 01 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 416 (1): 109–116.
Article history
Received:
May 15 2008
Revision Received:
June 25 2008
Accepted:
July 01 2008
Accepted Manuscript online:
July 01 2008
Citation
Yoshikuni Goto, Hiroe Tanji, Akira Hattori, Masafumi Tsujimoto; Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1. Biochem J 15 November 2008; 416 (1): 109–116. doi: https://doi.org/10.1042/BJ20080965
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