STRUCTURAL BASES FOR CATALYTIC EFFICIENCY AND SUBSTRATE SPECIFICITY

描述（由申请人提供）：酶的催化效率和底物特异性的基于结构的解释是难以捉摸的。该项目的目标包括：1)从结构上解释orotidine 5'-单磷酸脱羧酶（OMPDC）催化嘧啶核苷酸生物合成途径最后一步的非凡催化效率；2)表征d -核酮糖1,5-二磷酸羧化酶/加氧酶（RuBisCO）超家族的底物特异性和机制多样性。具体目标1侧重于活性位点残基在ompdc催化反应中基态不稳定（GSD）和过渡态稳定（TSS）中的作用。我们已经确定了反应中有中间产物，现在我们想了解中间产物产生和稳定的机理。我们将研究特定氨基酸残基在稳定中间体嘧啶环（TSS）中离域负电荷的作用，以及那些被认为参与破坏底物羧酸基（GSD）稳定的氨基酸残基。我们将与博士合作。Arthur Palmer，哥伦比亚，核磁共振结构和动力学研究；Jiali Gao博士，明尼苏达州，计算研究。特异性目标2侧重于在ompdc催化反应中，底物结合与催化作用耦合的结构机制。使用位点定向诱变和“两部分”底物类似物（亚磷酸盐阴离子和截断的奥罗替丁）的研究。John Richard和Tina Amyes， Buffalo)证明了远端磷酸基团协同脱羧。我们将研究伴随底物结合的构象变化，以确定特定残基在偶联结合催化中的作用。特异性目标3集中于表征RuBisCO超家族的机制多样性。这些结构表明，未表征的成员将是使用综合实验和计算方法进行功能预测的优秀候选者，该方法成功地识别了酰胺水解酶和烯醇化酶超家族中的新反应；我们将与加州大学旧金山分校的Matthew P. Jacobson博士合作，进行同源性建模和计算机库对接。随着新功能的发现，机制将被表征，从而可以描述功能和机制多样性的结构基础。这些研究将有助于1)描述OMPDC抑制剂设计的结构策略（超出当前项目的范围）；2)加强整合结构-功能-计算方法，以分配基因组计划中发现的未表征蛋白质的功能。公共卫生相关性：该项目侧重于利用基因组信息为基因组测序项目中发现的蛋白质分配功能以及建立生物功能的结构基础的重要生物医学问题。如果要为小分子干预（药物）设计新的靶点和实验方法，就必须知道所有蛋白质在生物体的分子、细胞和有机体功能中的作用。本项目利用orotidine 5'-单磷酸脱羧酶（OMPDC）和d -核酮糖1,5-二磷酸羧化酶/加氧酶（RuBisCO）的同源物，对生物功能的结构基础有了新的认识，该酶是RNA和DNA生物合成的必需酶。我们对ompdc催化反应的研究将为设计用作抗生素的抑制剂提供新的策略；我们对RuBisCO超家族的研究将促进新方法的发展，用于分配基因组计划中发现的未表征蛋白质的功能。

DESCRIPTION (provided by applicant): Structure-based explanations for the catalytic efficiencies and substrate specificities of enzymes are elusive. The goals of this project include 1) providing a structure-based explanation for the extraordinary catalytic efficiency of orotidine 5'-monophosphate decarboxylase (OMPDC) that catalyzes the final step in the pathway for pyrimidine nucleotide biosynthesis, and 2) characterizing the substrate specificity and mechanistic diversity in the D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) superfamily. Specific Aim 1 focuses on the roles of active site residues in both ground state destabilization (GSD) and transition state stabilization (TSS) in the OMPDC-catalyzed reaction. We have established that the reaction involves an intermediate, and we now want to understand the mechanisms by which the intermediate is generated and stabilized. We will examine the roles of specific amino acid residues in stabilizing negative charge that is delocalized in the pyrimidine ring of the intermediate (TSS) as well as those putatively involved in destabilizing the substrate carboxylate group (GSD). We will collaborate with Drs. Arthur Palmer, Columbia, for NMR studies of structure and dynamics and Dr. Jiali Gao, Minnesota, for computational studies. Specific Aim 2 focuses on the structural mechanism by which substrate binding is coupled to catalysis in the OMPDC-catalyzed reaction. Studies using both site-directed mutagenesis and "two part" substrate analogs (phosphite anion and a truncated orotidine, Drs. John Richard and Tina Amyes, Buffalo) demonstrate that the remote phosphate group synergizes decarboxylation. We will examine the conformational changes that accompany substrate binding to establish the roles of specific residues in coupling binding to catalysis. Specific Aim 3 is focused on characterizing mechanistic diversity in the RuBisCO superfamily. The structures suggest that the uncharacterized members will be excellent candidates for functional prediction using an integrated experimental and computational approach that successfully identified novel reactions in the amidohydrolase and enolase superfamilies; we will collaborate with Dr. Matthew P. Jacobson, UCSF, for both homology modeling and in silico library docking. As new functions are discovered, the mechanisms will be characterized so that the structural bases of functional and mechanistic diversity can be described. These studies will contribute to 1) delineating structural strategies for the design of inhibitors of OMPDC (beyond the scope of the current project); and 2) enhancing integrated structure-function-computation approaches for assigning the functions of uncharacterized proteins discovered in genome projects. PUBLIC HEALTH RELEVANCE: This project is focused on the important biomedical problem of exploiting genomic information to both assign functions to proteins discovered in genome sequencing projects and, also, to establish the structural bases for the biological functions. If new targets and experimental approaches are to be devised for small molecule intervention (drugs), the roles of all proteins involved in an organism's molecular, cellular, and organismal functions must be known. This project uses orotidine 5'-monophosphate decarboxylase (OMPDC), an essential enzyme in the biosynthesis of RNA and DNA, and homologues of D-ribulose 1,5-bisphosphate carbxoylase/oxygenase (RuBisCO), the ubiquitious enzyme that fixes CO2, to develop new knowledge about the structural basis for biological function. Our studies of OMPDC-catalyzed reaction will enable new strategies for the design of inhibitors for use as antibiotics; our studies of the RuBisCO superfamily will enhance the development of new approaches for assigning the functions of uncharacterized proteins discovered in genome projects.