An Investigation of the Chemical and Catalytic Mechanism of Pyruvate Carboxylase

描述(由申请人提供)： 丙酮酸羧基酶(PC)催化丙酮酸与HC03-反应生成草酰乙酸酯 在糖异生的第一步，在蛋白质的两个空间上不同的亚基上存在三磷酸腺苷和乙酰辅酶A。PC活性和调节的异常与II型糖尿病的发生有关。目前，我们对催化反应中发生的反应步骤、过渡态和基团转移步骤缺乏详细的了解。我们打算使用稳态动力学、同位素效应和核磁共振实验来获得有助于开发II型糖尿病和遗传性肥胖症潜在治疗方法的机制细节。 野生型和突变型PC的稳态动力学分析将被用来确定特定氨基酸在整个催化机制中的一般作用，这些氨基酸是基于Resti PC全酶的结构数据和序列同源性进行定点突变的。ID和2D核磁共振实验将被用来探索生物素修复基团的结构域间移动，以及在催化过程中生物素和丙酮酸的烯醇化反应机理。利用同位素比值质谱分析和内竞争或远程标记的方法测量单个反应步骤的动力学同位素效应，以探索过渡态和相关反应步骤的性质。稳态动力学分析与同位素效应和核磁共振实验相结合，将使我们能够对整个催化反应中发生的各个反应步骤获得宝贵的见解。 丙酮酸羧化酶是葡萄糖代谢途径中的重要调节酶。PC活性和葡萄糖刺激的胰岛素分泌之间的联系使其成为治疗II型糖尿病和遗传性肥胖的有吸引力的药物靶点。为了充分发挥PC作为药物靶点的潜力，拟议的研究将集中在从化学水平上了解PC如何催化糖异生的第一步。

DESCRIPTION (provided by applicant): Pyruvate carboxylase (PC) catalyzes the formation of oxaloacetate from pyruvate and HC03- in the presence of ATP and acetyl-CoA at two spatially distinct subunits of the protein in the first committed step of gluconeogenesis. Abnormalities in PC activity and regulation have been associated with the occurrence of Type II diabetes. Currently, we lack a detailed understanding of the reaction steps, transition states, and group transfer steps occurring in the catalytic reaction. We intend to use steady state kinetics, isotope effects, and NMR experiments to obtain mechanistic details beneficial for the development of potential treatments for Type II diabetes and genetic obesity. Steady state kinetic analysis of wild type and mutant forms of PC will be used to determine the general roles of specific amino acids, targeted for site-directed mutagenesis based on structural data for the R. etii PC holoenzyme and sequence homology, in the overall catalytic mechanism. ID and 2D NMR experiments will be used to probe the interdomain movement of the biotin prosthetic group and explore biotin and pyruvate enolization during the catalytic mechanism. Isotope ratio mass spectrometric analysis and the internal competition or remote labeling methods will be used to measure the kinetic isotope effects of individual reaction steps to probe the nature of transition states and relative reaction steps. The steady state kinetic analysis coupled with isotope effect and NMR experiments will allow us to gain invaluable insight into the individual reaction steps occurring throughout the catalytic reaction. Pyruvate carboxylase is an important regulatory enzyme in the metabolic pathway for glucose production. The link between PC activity and glucose-stimulated insulin secretion makes it an attractive drug target for the treatment of Type II diabetes and genetic obesity. In order to fully realize PC's potential as a drug target, the research proposed will focus on understanding, at the chemical level, how PC catalyzes the first step in gluconeogenesis.