STRUCTURAL BASES FOR CATALYTIC EFFICIENCY AND SUBSTRATE SPECIFICITY

描述（由申请人提供）：对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）为乳清酸核苷5 '-单磷酸脱羧酶（OMPDC）的非凡催化效率提供基于结构的解释，该酶催化嘧啶核苷酸生物合成途径中的最后一步; 2）表征D-核酮糖1，5-二磷酸羧化酶/加氧酶（RuBisCO）超家族中的底物特异性和机制多样性。具体目标1侧重于OMPDC催化反应中活性位点残基在基态不稳定（GSD）和过渡态稳定（TSS）中的作用。我们已经确定反应涉及中间体，现在我们想了解中间体产生和稳定的机制。我们将研究特定氨基酸残基在稳定中间体（TSS）的嘧啶环中离域的负电荷中的作用，以及参与使底物羧酸基团（GSD）不稳定的那些puerides。我们将与哥伦比亚的亚瑟帕尔默博士合作进行结构和动力学的核磁共振研究，并与明尼苏达州的高佳丽博士合作进行计算研究。具体目标2侧重于底物结合与OMPDC催化反应中的催化作用相结合的结构机制。使用定点诱变和“两部分”底物类似物（亚磷酸根阴离子和截短乳清酸苷，Drs. John Richard和Tina Amyes，布法罗）的研究表明，远程磷酸基团协同脱羧作用。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。具体目标3专注于表征RuBisCO超家族中的机制多样性。这些结构表明，未表征的成员将是使用集成的实验和计算方法进行功能预测的优秀候选者，该方法成功地确定了酰胺水解酶和烯醇化酶超家族中的新反应;我们将与UCSF的Matthew P. Jacobson博士合作进行同源建模和计算机库对接。随着新功能的发现，机制将被表征，以便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）描绘用于设计OMPDC抑制剂的结构策略（超出当前项目的范围）;和2）增强用于分配基因组计划中发现的未表征蛋白质的功能的集成结构-功能-计算方法。公共卫生相关性：该项目的重点是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这一重要的生物医学问题。如果要为小分子干预（药物）设计新的靶点和实验方法，则必须知道参与生物体分子、细胞和生物体功能的所有蛋白质的作用。该项目使用乳清酸核苷5 '-单磷酸脱羧酶（OMPDC），一种RNA和DNA生物合成中的必需酶，以及D-核酮糖1，5-二磷酸羧化酶/加氧酶（RuBisCO）的同系物，一种固定CO2的泛素酶，以开发有关生物功能结构基础的新知识。我们对OMPDC催化反应的研究将使设计用作抗生素的抑制剂的新策略成为可能;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.