Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia
Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia
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DOI：
10.1021/bi991570i
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发表时间：
2000-01-11
期刊：
影响因子：
2.9
通讯作者：
Zhang, ZY
中科院分区：
文献类型：
--
作者：
Hoff, RH;Hengge, AC;Zhang, ZY
蛋白质酪氨酸磷酸酶(PTPase)中的一般酸催化是由保守的Asp残基完成的，它是通过与底物结合时发生的柔性环的移动而进入催化位置的，利用耶尔森菌的PTPase，我们研究了两个保守残基的突变对一般酸催化的影响，这两个保守残基是这种构象变化所必需的。残基Trp354处于环路的铰链上：Arg409与底物的磷酰基形成氢键和离子相互作用。Trp354突变为Phe和Ala，Arg409残基突变为Lys和Ala。用稳态动力学和重原子同位素效应研究了这四种突变酶与底物对硝基苯磷酸的作用，结果表明，铰链残基Trp354突变为ALA完全丧失了一般的酸催化作用。在Phe突变体中，普通酸催化是部分有效的，但质子只在过渡态部分转移，与天然酶质子转移到离开基团几乎完全不同，Arp409到Lys的突变对K-m影响很小，而在ala突变体中，这一参数增加了30倍，R409K和R409A的k(CAT)值比天然酶低约4个数量级。一般的酸催化由于Lys突变而不起作用，但在催化过程中部分质子转移仍然发生在Ala突变体中。对这些突变对柔性环运动的不同影响进行了结构解释，使一般的酸催化成为可能。
General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate, With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop: and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate, The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete, Mutation of Arp409 to Lys has a minimal effect on the K-m, while this parameter is increased 30-fold in the Ala mutant, The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.