Mechanisms and Function in HAD Phosphotransferases

描述（由申请人提供）：卤代烷酸脱卤酶超家族（HADSF）是最大和最普遍的酶家族之一，在从细菌到人类的生物体中有3000多个成员。拟议的研究将使用稳态和瞬态动力学、x射线结构测定和生物信息学来确定选定的具有生物医学重要性的HADSF磷酸转移酶的催化和底物识别机制。在Aim 1中，将通过测量催化循环各个步骤的速率常数，使用野生型和位点定向突变体识别稳定磷烷中间体的残基，确定同步帽闭合和酸/碱催化的机制，以及测试帽结构域闭合在磷烷形成中的作用来定义β -磷酸葡糖化酶中磷酸化转移的催化机制。在Aim 1的第2部分中，将确定人类α -磷酸腺苷异变酶的结构，并发现与先天性糖基化疾病相关的野生型酶和突变体在底物识别、酶磷酸化、底物重定向、构象改变和过渡状态稳定中的单个残基的作用。目的2针对三个HADSF亚家族中两个的底物识别机制。在第1部分中，我们将使用溶剂笼法对PDB中与HADSF型MB磷酸酶对应的孤儿结构进行功能分配，以确定底物筛选的引线。糖磷酸酶在单一物种内的明显功能冗余将通过确定同源物的底物范围来探测。在第二部分中，III型亚家族细菌酶N-酰基神经氨酸-9-磷酸磷酸酶和2-酮-3-脱氧-d -甘露糖-八-磷酸磷酸酶将从结构和底物混杂性方面进行表征，以确定底物特异性决定因素，并发现酸碱催化和底物诱导配合是否有效。

DESCRIPTION (provided by applicant): The haloalkanoate dehalogenase superfamily (HADSF) is one of the largest and most ubiquitous enzyme families, with over 3,000 members in organisms ranging from bacteria to humans. The proposed studies will define, using steady-state and transient-state kinetics, X-ray structure determination, and bioinformatics, the mechanisms of catalysis and substrate recognition in selected HADSF phosphotransferases of biomedical importance. In Aim 1, the mechanism of catalysis of phosphoryl transfer in beta-phosphoglucomutase will be defined by measuring rate constants for individual steps of the catalytic cycle, identifying residues that stabilize the phosphorane intermediate using wild type and site- directed mutants, determining the mechanism of synchronizing cap closure and acid/base catalysis, and testing the role of cap domain closure in phosphorane formation. In part 2 of Aim 1, the structure of human alpha-phosphomannomutase will be determined and the roles of individual residues in substrate recognition, enzyme phosphorylation, substrate reorientation, conformational changes, and transition-state stabilization will be found for the wild-type enzyme and mutants clinically correlated with congenital disorders of glycosylation. Aim 2 targets the mechanisms of substrate recognition in two of the three HADSF subfamilies. In part 1, we will provide functional assignment to orphaned structures from the PDB corresponding to HADSF type MB phosphatases using a solvent cage method to identify leads for substrate screening. The apparent functional redundancy in sugar phosphatases within a single species will be probed by determining substrate range in homologs. In part 2, the Type III subfamily bacterial enzymes N- acyl-neuraminate-9-phosphate phosphatase and 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate phosphatase will be characterized structurally and in terms of substrate promiscuity, in order to define the substrate specificity determinants and find if acid/base catalysis and substrate induced fit are operative.