STRUCTURAL AND MECHANISTIC STUDIES OF PHOSPHONATASE

描述（申请人摘要）：本申请中拟定的研究 新的拨款申请将审查结构和行动机制， 酶磷酸酶和扩展结构和机制的研究，以其他 卤酸脱卤酶（HAD）超家族成员。每种酶 该超家族使用保守的Asp残基形成 酰基磷酸-酶中间体或烷基酯-酶中间体。这 化学是由共同的结构支架支撑的。磷酸酶催化 膦酰基乙醛（P-Ald）水解成乙醛， 正磷酸盐与2-氨基乙基膦酸酯转氨酶结合， 磷酸酶在用于回收P的两步生物降解途径中起作用， N和C来自无处不在的天然膦酸酯，2-氨基乙基膦酸酯。 尽管已知的生物活性范围广泛， 和合成膦酸盐，膦酸盐代谢的酶学很差， 表征了这些研究的目的是了解 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测试机械模型 基于最近确定的磷酸酶X射线结构（艾伦 实验室）和以前的机制研究（Dunaway-Mariano实验室）。 将使用定点突变结合瞬时动力学分析 来测试模型的化学步骤。晶体结构 对使用底物形成的死端复合物进行测定 类似物、酶突变体和化学修饰的酶将用于 捕获所提出的反应中间体的结构。第二组 建议的实验将研究HAD的活性位点多样化 酶超家族磷酸酶催化以下反应的能力 其他家庭成员将被确定，蛋白质工程将被用来 交换两个家庭成员的催化活性。为了进一步探索催化剂 超家族的可塑性， β-磷酸葡萄糖变位酶，一种来自HAD家族的磷酸转移酶， 考察这些研究的目的是了解 酶超家族的活性位点已被改造成催化C-X、P-O和C-P 在各种不同的衬底结构中的键断裂。

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.