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首页> 外文期刊>Journal of Molecular Biology >Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase
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Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

机译：钨酸盐作为碱性磷酸酶催化的过渡状态模拟

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摘要

The catalytic mechanisms underlying Escherichia coli alkaline phosphatase's (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein. The crystal structure of tungstate-bound alkaline phosphatase provides evidence for a covalent adduct model and further shows that the ligand adopts trigonal bipyramidal geometry, which is infrequently observed for tungstate in small molecules and other active sites but mirrors the geometry of the presumed phosphoryl transfer transition state. The AP active site is known to stabilize another oxyanion, vanadate, in trigonal bipyramidal geometry, but the extent to which binding of either ligand reproduces the energetics of the transition state cannot be deduced from structural inspection alone. To test for transition state analog behavior, we determined the relationship between catalytic activity and affinity for tungstate and vanadate for a series of 20 AP variants. Affinity and activity were highly correlated for tungstate (r(2) = 0.89) but not vanadate (r(2) = 0.23), indicating that the tungstate circle AP complex may better mimic this enzyme's transition state properties. The results herein suggest that tungstate will be a valuable tool for further dissecting AP catalysis and may prove helpful in mechanistic studies of other phosphoryl transfer enzymes. (C) 2016 Elsevier Ltd. All rights reserved.
机译：大肠杆菌碱性磷酸酶（AP）显着速率增强的催化机制已被广泛探测。过去的工作表明，虽然丝氨酸核酸（SER102）静电地排斥产物磷酸盐，但在Ser102的存在下，将另一种氧气钨酸盐，钨酸盐更强烈地结合。这些结果预测了丝氨酸亲核官和钨酸酮之间的共价键，我们在此测试的模型。钨酸盐碱性磷酸酶的晶体结构提供了共价加合物模型的证据，进一步表明，配体采用三角形的双吡酰胺几何形状，该几何形状不经常观察到小分子和其他活性位点，但反映了推定的磷磷转移过渡的几何形状状态。已知AP活性位点在三角形双吡酰胺几何形状中稳定另一种氧气，钒酸盐，但是单独的结构检查不能推导出转换状态的高能量的程度。为了测试过渡状态模拟行为，我们确定催化活性与钨酸盐和钒酸盐之间的关系之间的关系，用于一系列20个AP变体。亲和力和活性对于钨酸盐（R（2）= 0.89）高度相关，但不钒酸盐（R（2）= 0.23），表明钨酸酯圈AP复合物可以更好地模仿该酶的过渡状态性质。本文的结果表明，钨酸酮是一种有价值的工具，用于进一步解剖AP催化，并且可以有助于其他磷酸转移酶的机械研究。 （c）2016 Elsevier有限公司保留所有权利。

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