Catalytic function of the conserved hydroxyl group in the protein tyrosine phosphatase signature motif.
Catalytic function of the conserved hydroxyl group in the protein tyrosine phosphatase signature motif.
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DOI：
10.1021/bi00050a020
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发表时间：
1995-12
期刊：
影响因子：
2.9
通讯作者：
Z. Zhang;B. Palfey;Li Wu;Y. Zhao
中科院分区：
文献类型：
--
作者：
Z. Zhang;B. Palfey;Li Wu;Y. Zhao
用耶尔森菌蛋白酪氨酸磷酸酶（PTPase）观察爆发动力学。这为磷酸酶机制提供了直接的动力学证据，并表明磷酸酶中间体的分解是限速步骤。爆发动力学是研究PTPase催化机理的有力工具，因为活性位点残基的功能作用可以通过研究它们对与中间体形成和分解相关的单个基本步骤的影响来评估。为了研究Thr410（一个存在于PTPase特征基序中的保守残基）的作用，通过位点定向突变将该残基改变为丝氨酸和丙氨酸。这些突变的影响，在以对硝基苯基磷酸（pNPP）为底物的稳态和预稳态动力学实验中观察到，表明Thr410的羟基直接参与催化。残基410处的羟基在促进磷酸酶中间体的分解中起重要作用。
Burst kinetics is observed with the Yersinia protein tyrosine phosphatase (PTPase). This provides direct kinetic evidence for a phosphoenzyme mechanism and suggests that the breakdown of the phosphoenzyme intermediate is the rate-limiting step. Burst kinetics is a powerful tool for mechanistic studies of PTPase catalysis since functional roles of active site residues can be evaluated by studying their effects on the individual elementary steps associated with the formation and the breakdown of the intermediate. In order to investigate the role of Thr410, a conserved residue that is present in the PTPase signature motif, this residue was altered by site-directed mutagenesis to serine and alanine. The effects of these mutations, as observed in both steady-state and pre-steady-state kinetic experiments with p-nitrophenyl phosphate (pNPP) as a substrate, demonstrated that the hydroxyl group of Thr410 is directly involved in catalysis. The hydroxyl group at residue 410 plays an important role in facilitating the breakdown of the phosphoenzyme intermediate.