Reactivity of alcohols toward the phosphoenzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction: probing the transition state of the dephosphorylation step.
Reactivity of alcohols toward the phosphoenzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction: probing the transition state of the dephosphorylation step.
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DOI：
10.1021/bi960471r
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发表时间：
1996-09
期刊：
影响因子：
2.9
通讯作者：
Y. Zhao;Z. Zhang
中科院分区：
文献类型：
--
作者：
Y. Zhao;Z. Zhang
在溶液中，磷酸单酯通过高度解离的机制水解，该机制涉及“松散的”或“爆炸的”偏磷酸盐样过渡状态，其中与进入的亲核试剂的键形成最小，并且磷和离去基团之间的键断裂是显著的。为了更好地理解蛋白酪氨酸磷酸酶（PTH 4）如何影响催化作用，确定酶的过渡态的性质是很重要的。PTPases通过两步机制催化磷酸单酯的水解，该机制通过磷酸酶中间体（E-P）进行。广泛的重原子动力学同位素效应和离去基团依赖性的研究提供了洞察过渡态的性质的第一步（E-P形成）的PTH 4反应。本文通过改变醇的碱性对其与E-P反应活性的影响，探讨了低M（r）PTPase-catalytic dephosphorylation反应的过渡态，测得醇与E-P反应的Brønsted β nu值为0.14，表明酶促反应的过渡态是高度解离的，与无催化的溶液反应类似。我们表明，保守的羟基在PTARY签名基序主要参与E-P去磷酸化步骤。我们进一步证明，羟基基团的消除使E-P去磷酸化的过渡态解离性降低，这表明在PTK活性位点中的羟基基团的主要功能是促进E-P通过解离途径并稳定解离过渡态。
In solution phosphate monoesters hydrolyze via a highly dissociative mechanism involving a "loose" or "exploded" metaphosphate-like transition state where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To better understand how protein-tyrosine phosphatase (PTPase) effects catalysis, it is important to determine the nature of the enzymic transition state. PTPases catalyze the hydrolysis of phosphate monoesters by a two-step mechanism that proceeds through a phosphoenzyme intermediate (E-P). Extensive heavy atom kinetic isotope effect and leaving group dependency studies have provided insights into the nature of the transition state for the first step (E-P formation) of the PTPase reaction. In this paper we have probed the transition state for the low M(r) PTPase-catalyzed dephosphorylation step by studying the effect of changing the alcohol basicity on its reactivity toward E-P. The Brønsted beta nu value for the reactions of alcohols and E-P is determined to be 0.14, which indicates that the enzymic transition state is highly dissociative and similar to that in uncatalyzed solution reactions. We show that the conserved hydroxyl group in the PTPase signature motif is primarily involved in the E-P dephosphorylation step. We further demonstrate that elimination of the hydroxyl group renders the transition state for E-P dephosphorylation less dissociative, suggesting that the main function of the hydroxyl group in the PTPase active site is to promote the E-P going through a dissociative pathway and to stabilize the dissociative transition state.