Engineering e-coli alkaline phosphatase yields changes of catalytic activity, thermal stability and phosphate inhibition
文献类型：期刊论文
| 作者 | Zhang, XE; Zhou, YH; Zhang, ZP; Xu, HF; Shao, WH; Cass, AEG |
| 刊名 | Biocatalysis and biotransformation
 |
| 出版日期 | 2002 |
| 卷号 | 20期号:6页码:381-389 |
| 关键词 | Alkaline phosphatase Over-lap pcr Site-directed mutagenesis Catalytic activity Phosphate inhibition |
| ISSN号 | 1024-2422 |
| DOI | 10.1080/1024242021000058667 |
| 通讯作者 | Zhang, xe() |
| 英文摘要 | To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of escherichia coli alkaline phosphatase (eap), two single mutants d101s (asp 101 --> ser) and r166k (arg 166 --> lys) and a double mutant d101s/r166k of eap were generated through site-directed mutagenesis based on over-lap pcr method. their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. in the presence of inorganic phosphate acceptor, 1 m diethanolamine buffer, the k(cat) for d101s mutant enzyme increased 10-fold compared to that of wild-type eap. the mutant r166k has a 2-fold decrease of k(cat) relative to the wild-type eap, but the double mutant d101s/r166k was in the middle of them, indicative of an additive effect of these two mutations. on the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of k-m values. all three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. the analysis of the kinetic data suggests that (1) the d101s mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the r166k mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. additionally, pre-steady-state kinetics of d101s and d101s/r166k mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type eap, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent e-pi complex to the hydrolysis of covalent e-pi complex for these two mutants. |
| WOS关键词 | ESCHERICHIA-COLI ; SITE ; MECHANISM ; MUTAGENESIS ; RESOLUTION ; ENZYMES |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS类目 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000179319500001 |
| 出版者 | TAYLOR & FRANCIS LTD |
| URI标识 | http://www.irgrid.ac.cn/handle/1471x/2376108 |
| 专题 | 武汉病毒研究所 |
| 通讯作者 | Zhang, XE |
| 作者单位 | 1.Chinese Acad Sci, Wuhan Inst Virol, Wuhan 430071, Peoples R China 2.Univ London Imperial Coll Sci Technol & Med, Dept Biol Sci, London SW7 2AY, England |
| 推荐引用方式 GB/T 7714 | Zhang, XE,Zhou, YH,Zhang, ZP,et al. Engineering e-coli alkaline phosphatase yields changes of catalytic activity, thermal stability and phosphate inhibition[J]. Biocatalysis and biotransformation,2002,20(6):381-389. |
| APA | Zhang, XE,Zhou, YH,Zhang, ZP,Xu, HF,Shao, WH,&Cass, AEG.(2002).Engineering e-coli alkaline phosphatase yields changes of catalytic activity, thermal stability and phosphate inhibition.Biocatalysis and biotransformation,20(6),381-389. |
| MLA | Zhang, XE,et al."Engineering e-coli alkaline phosphatase yields changes of catalytic activity, thermal stability and phosphate inhibition".Biocatalysis and biotransformation 20.6(2002):381-389. |
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来源：武汉病毒研究所
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