STRUCTURAL AND MECHANISTIC STUDIES OF PHOSPHONATASE

描述(申请人摘要)：本研究拟进行的研究 新的拨款申请将审查基金的结构和运作机制 酶磷酸酶，并将结构和机理研究扩展到其他 卤酸脱卤酶(HAD)酶超家族成员。每一种酶 这个超家族使用保守的Asp残基形成一个 酰基磷酸-酶中间体或烷基酯-酶中间体。这 化学是由一个共同的结构支架支撑的。磷酸酶催化 膦乙醛(P-Ald)的水解制乙醛 正磷酸盐。与2-氨基乙基膦转氨酶结合， 磷酸酶在用于循环P的两步生物降解途径中发挥作用， N和C来自普遍存在的天然膦酸根2-氨基乙基膦酸根。 尽管已知的广泛的生物活性与天然的 和合成的磷酸盐，磷酸盐代谢的酶学很差 特色化的。这些研究的目的是为了了解 以磷酸酶为模型的酶催化C-P键断裂过程 系统。提出的第一组实验将检验机械模型。 基于最近确定的磷酸酶X-射线结构(Allen 实验室)和以前的机械研究(达纳韦-马里亚诺实验室)。 将使用定点突变和瞬时动力学分析相结合的方法 来测试模型的化学步骤。晶体结构 使用底物形成的死端络合物的测定 类似物、酶突变体和化学修饰的酶将用于 捕获建议的反应中间体的结构。第二套 建议进行的试验将会研究民政事务总署用地多元化的问题。 酶超家族。磷酸酶对苯丙氨酸反应的催化能力 将确定其他家庭成员，并将使用蛋白质工程来 两个家族成员的交换催化活性。进一步探索催化作用 超家族的可塑性、结构和机制 来自HAD家族的磷酸转移酶-β-磷酸葡萄糖变位酶将是 检查过了。这些研究的目标是了解人类是如何 酶超家族活性中心适用于催化C-X、P-O和C-P 在各种不同的底物结构中发生键断裂。

DESCRIPTION ( applicant's abstract): The studies proposed in this new grant application will examine the structure and mechanism of action of the enzyme phosphonatase and extend structural and mechanistic studies to other members of the haloacid dehalogenase (HAD) enzyme superfamily. Each enzyme of this superfamily uses a conserved Asp residue to form either an acylphosphate-enzyme intermediate or an alkyl ester-enzyme intermediate. This chemistry is supported by a common structural scaffold. Phosphonatase catalyzes the hydrolysis of phosphonoacetaldehyde (P-Ald) to acetaldehyde and orthophosphate. In conjunction with 2-aminoethylphosphonate transaminase, phosphonatase functions in a two-step biodegradative pathway used to recycle P, N, and C from the ubiquitous natural phosphonate, 2-aminoethylphosphonate. Despite the wide range of known biological activities associated with natural and synthetic phosphonates, the enzymology of phosphonate metabolism is poorly characterized. The goal of these studies is to derive an understanding of the process of enzyme catalyzed C-P bond cleavage using phosphonatase as the model system. The first set of experiments proposed will test mechanistic models based on the recently determined phosphonatase X-ray structure (Allen laboratory) and on previous mechanistic studies (Dunaway-Mariano laboratory). Site-directed mutagenesis coupled with transient kinetic analysis will be used to test the chemical steps of the models. Crystallographic structure determinations carried out on dead-end complexes formed using substrate analogues, enzyme mutants, and chemically modified enzymes will be used to capture the structures of proposed reaction intermediates. The second set of experiments proposed will examine the active-site diversification of the HAD enzyme superfamily. The ability of phosphonatase to catalyze the reactions of other family members will be determined and protein engineering will be used to swap catalytic activities of two family members. To further probe the catalytic plasticity of the superfamily, the structure and mechanism of beta-phosphoglucomutase, a phosphotransferase from the HAD family will be examined. The goal of these studies is to derive an understanding of how the enzyme superfamily active site has been adapted to catalyze C-X, P-O and C-P bond cleavage in a variety of different substrate structures.