Mechanisms and Function in HAD Phosphotransferases

描述(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，在从细菌到人类的各种生物体中有超过3,000个成员。这项拟议的研究将利用稳态和瞬时动力学、X射线结构测定和生物信息学，确定具有生物医学意义的特定HADSF磷酸转移酶的催化和底物识别机制。在目标1中，通过测量催化循环各个步骤的速率常数，利用野生型和定点突变体鉴定稳定磷烷中间体的残基，确定同步封帽和酸碱催化的机制，以及测试帽结构域关闭在磷烷形成中的作用，来定义β-磷酸葡萄糖变位酶中磷酸转移的催化机制。在目标1的第二部分中，将确定人α-磷酸甘露聚糖酶的结构，并将发现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，这些酶和突变体临床上与先天性糖基化障碍相关。目的研究HADSF三个亚家族中的两个亚家族的底物识别机制。在第一部分中，我们将使用溶剂笼法从与HADSF型MB磷酸酶相对应的PDB中为孤儿结构提供功能分配，以确定底物筛选的线索。将通过测定同系物中的底物范围来探索单一物种中糖磷酸酶的明显功能冗余。在第二部分中，我们将对III型亚家族细菌酶N-酰神经氨酸-9-磷酸磷酸酶和2-keto-3-deoxy-D-manno-octulosonate-8-phosphate磷酸酶的结构和底物杂交性进行表征，以确定底物特异性决定因素，并找出酸碱催化和底物诱导FIT是否起作用。

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.