Mechanisms of Acyl, Phosphoryl and Sulfuryl Transfer

描述(申请人提供)：蛋白质磷酸化和去磷酸化是细胞用来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是磷酸化的。磷酸基转移在生物学上的重要性和普遍性促使人们在理解催化这些反应的酶的机制方面做出了相当大的努力。尽管磷酸酯的非催化水解是已知最慢的反应之一，但在生物途径中普遍存在着磷酸酶。 这个项目将探索几个可能有助于蛋白质酪氨酸磷酸酶(PTPase)极高催化效率的潜在因素。将使用[31]P核磁共振中的[18]O同位素位移来研究溶液中的去溶和反离子效应，并通过测量底物与催化失活的磷酸酶结合的平衡同位素效应来评估局部环境效应破坏离子磷酸酯底物的稳定性从而有助于酶催化的可能性。另外，将使用几种动力学和热力学方法来研究蛋白质构象变化的作用，这是PTPase共同的催化机制的一部分。蛋白质构象异构化可能与化学催化相耦合，从而是催化能力的来源的假设将得到验证。最后，我们将对一种嗜热生物的PTPase进行机理研究。磷酸单酯的水解表现出异常高的温度依赖性(非催化的烷基磷酸水解率每15摄氏度增加约30倍)。此外，与其他生物相关的水解反应的情况相比，活化的熵是正的(有利的)。因此，克服熵不能成为磷酸酶催化效率的重要来源。这些因素使嗜热生物磷酸酶的酶学本身很有趣，也是研究热力学对催化的贡献的工具，这在高温和低温生物中可能是不同的。

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.