Mechanisms of Acyl, Phosphoryl and Sulfuryl Transfer

描述（由申请人提供）：蛋白质磷酸化和去磷酸化构成细胞控制生物过程的基本机制;据估计，所有细胞蛋白质的三分之一被磷酸化。磷酰基转移的生物学重要性和普遍性激发了相当大的努力，以了解催化这些反应的酶的机制。尽管磷酸酯的非催化水解是已知的最慢的反应之一，但磷酸酶在生物途径中的普遍性已经出现。 该项目将探索可能有助于蛋白酪氨酸磷酸酶（PTPases）极高催化效率的几个潜在因素。潜在的局部环境影响，使离子型磷酸酯底物不稳定，从而有助于酶催化，将使用[18]O同位素位移[31]P NMR研究溶液中的去溶剂化和反溶剂化效应，并通过测量平衡同位素效应对底物结合催化禁用磷酸酶进行评估。另外，蛋白质构象变化的作用，这是一个共同的PTPases的催化机制的一部分，将进行研究，使用几种动力学和热力学方法。蛋白质构象异构化可能与化学催化作用相结合，因此是催化能力的一个来源，这一假设将得到验证。最后，将进行嗜热生物的PTODN的机制研究。磷酸单酯水解显示出异常高的温度依赖性（每15 ℃，未催化的磷酸烷基酯水解的速率增加约30倍）。此外，与其他生物相关的水解反应的情况相比，活化熵是正的（有利的）。因此，克服熵不能是磷酸酶催化效率的重要来源。这些因素使得嗜热生物的磷酸酶的酶学在它们自己的权利中令人感兴趣，以及研究对催化的热力学贡献的载体，这在高温和低温生物中可能不同。

DESCRIPTION (provided by applicant): Protein phosphorylation and dephosphorylation constitute a fundamental mechanism used by cells to control biological processes; it has been estimated that a third of all cellular proteins are phosphorylated. The biological importance and ubiquity of phosphoryl transfer has motivated considerable effort toward understanding the mechanisms of the enzymes that catalyze these reactions. The ubiquity of phosphatases in biological pathways has arisen in spite of the fact that the uncatalyzed hydrolysis of a phosphate ester is among the slowest reactions known. This project will explore several potential factors that may contribute to the extremely high catalytic efficiencies of protein-tyrosine phosphatases (PTPases). The potential for local environmental effects to destabilize the ionic phosphate ester substrates, and thus to contribute to enzymatic catalysis, will be evaluated using [18]O isotopic shifts in [31]P NMR to study desolvation and counterion effects in solution, and by measurement of equilibrium isotope effects on binding of substrates to catalytically disabled phosphatases. Separately, the role of a protein conformational change that is a part of the catalytic mechanism common to PTPases will be studied, using several kinetic and thermodynamic methods. The hypothesis that protein conformational isomerization may be coupled to chemical catalysis, and thus is a source of catalytic power, will be tested. Finally, a mechanistic study of a PTPase from a thermophilic organism will be carried out. Phosphate monoester hydrolysis shows an unusually high temperature dependence (the rate of uncatalyzed alkyl phosphate hydrolysis increases approximately 30-fold for each 15 degrees C). Furthermore, the entropy of activation is positive (favorable) in contrast to the situation with other biologically relevant hydrolytic reactions. Thus, overcoming entropy cannot be a significant source of catalytic efficiency for phosphatases. These factors make the enzymology of phosphatases from thermophilic organisms interesting in their own right, as well as a vehicle to study the thermodynamic contributions to catalysis, which may differ in high-temperature and low-temperature organisms.