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超过3，000个成员。拟议的研究将定义，使用稳态和瞬态动力学，X-射线结构测定和生物信息学，在选定的HADSF磷酸转移酶的生物医学重要性的催化和底物识别的机制。在目的1中，将通过测量催化循环的各个步骤的速率常数，使用野生型和定点突变体鉴定稳定正膦中间体的残基，确定同步帽闭合和酸/碱催化的机制，并测试帽结构域闭合在正膦形成中的作用，来定义β-磷酸葡萄糖变位酶中磷酰基转移的催化机制。在目标1的第2部分中，将确定人α-磷酸甘露变位酶的结构，并将发现野生型酶和与先天性糖基化疾病临床相关的突变体的单个残基在底物识别、酶磷酸化、底物重定向、构象变化和过渡态稳定中的作用。目的2针对三个HADSF亚家族中的两个的底物识别机制。在第1部分中，我们将提供从PDB对应于HADSF型MB磷酸酶的孤儿结构的功能分配，使用溶剂笼方法来识别用于底物筛选的引线。通过确定同源物中的底物范围来探测单个物种内糖磷酸酶的明显功能冗余。在第2部分中，III型亚家族细菌酶N-酰基-神经氨酸-9-磷酸酶和2-酮-3-脱氧-D-甘露-辛酮糖酸-8-磷酸酶将在结构上和底物混杂方面进行表征，以定义底物特异性决定簇并发现酸/碱催化和底物诱导的适合是否有效。

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.