論文 - 詳細
| RRC ID | 28646 |
|---|---|
| 著者 | Fukasawa KM, Hirose J, Hata T, Ono Y. |
| タイトル | Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B. |
| ジャーナル | Biochemistry |
| Abstract |
Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain. |
| 巻・号 | 45(38) |
| ページ | 11425-31 |
| 公開日 | 2006-9-26 |
| DOI | 10.1021/bi0604577 |
| PMID | 16981702 |
| MeSH | Alanine / metabolism Amino Acid Sequence Amino Acid Substitution Aminopeptidases / chemistry* Aminopeptidases / isolation & purification Aminopeptidases / metabolism* Animals Arginine / analogs & derivatives Arginine / chemistry Arginine / metabolism Aspartic Acid / metabolism* Epoxide Hydrolases / chemistry Epoxide Hydrolases / metabolism Glutamic Acid / metabolism Hydrolysis Kinetics Models, Biological Molecular Sequence Data Mutagenesis, Site-Directed Mutant Proteins / chemistry Mutant Proteins / metabolism Protein Binding Rats Recombinant Proteins / chemistry Recombinant Proteins / isolation & purification Sequence Alignment Structure-Activity Relationship Substrate Specificity |
| IF | 2.865 |
| 引用数 | 14 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Wikipedia |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | pGEXRApB (RDB07718) |