噬菌体T5脱氧核苷单磷酸激酶活性中心的定点诱变研究。

Bioorganicheskaia khimiia Pub Date : 2013-11-01
G V Mikulinskaia, S A Taran, Iu S Skoblov, S A Feofanov
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引用次数: 0

摘要

根据噬菌体T5脱氧核糖核苷单磷酸激酶活性中心的计算机模型,确定了该酶活性所必需的氨基酸残基。通过定点诱变,在大肠杆菌中克隆并表达了该基因,并将保守活性中心残基S13A、D16N、T17N、T17S、R130K、K131E、Q134A、G137A、T138A、W150F、W150A、D170N、R172I、E176Q进行了单氨基酸替换。利用离子交换和亲和层析步骤纯化突变体的电泳均质制剂。测定这些酶对磷酸基天然受体(dAMP, dCMP, dGMP, dTMP)的特异性酶活性表明,nmp结合域的带电残基(即R130, R172, D170和e176)的取代导致酶活性几乎完全丧失。结果表明,羟基在17位的存在对催化活性也很重要。根据比活性的变化,我们推测130位和172位的精氨酸残基参与了供体γ-磷酰基和受体α-磷酰基的结合。此外,atp结合位点(P-loop) 16位的天冬氨酸可能有助于受体的结合,首先是dTMP。部分活性突变体G137A、T138A、T17N、Q134A、S13A、D16N对不同底物的酶活性下降不均匀,表明不同底物的结合有不同的氨基酸残基参与。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
[Investigation of active center of deoxynucleoside monophosphate kinase of bacteriophage T5 by site-directed mutagenesis].

Based on the computer model of active center of bacteriophage T5 deoxyribonucleoside monophosphate kinase amino acid residues essential for the enzyme activity were determined. As the result of site-directed mutagenesis, cloning and expression of the gene in E. coli series of proteins were obtained with single amino acid substitutions of conservative active center residues--S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, E176Q. Electrophoretically homogeneous preparations of mutant forms were purified using ion exchange and affinity chromatographic steps. Measuring of the specific enzyme activities of these enzymes for the natural acceptors of phosphoryl group (dAMP, dCMP, dGMP, dTMP) revealed that substitutions of charged residues of NMP-binding domain-namely, R130, R172, D170 and E176-lead to almost complete loss of enzyme activity. It was shown that presence of OH-group at position 17 is also important for catalytic activity. Based on the changes in specific activities we suppose that arginine residues at positions 130 and 172 participate in binding of γ-phosphoryl of donor and α-phosphoryl of acceptor. Also, aspartic acid at 16 position of ATP-binding site (P-loop) probably assists in the binding of acceptor, first of all dTMP. Unequal decrease in enzyme activities for different substrates of partially active mutants--G137A, T138A, T17N, Q134A, S13A, D16N--indicate that in the binding of various substrates different amino acid residues take part.

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