An Investigation of the Chemical and Catalytic Mechanism of Pyruvate Carboxylase

描述（由申请人提供）： 丙酮酸羧化酶（PC）催化从丙酮酸和HCO 3-形成草酰乙酸。 在糖异生的第一个关键步骤中，蛋白质的两个空间不同亚基上存在ATP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，我们缺乏对催化反应中发生的反应步骤、过渡态和基团转移步骤的详细了解。我们打算使用稳态动力学，同位素效应，和NMR实验，以获得有利于II型糖尿病和遗传性肥胖症的潜在治疗的发展机制的细节。 PC的野生型和突变形式的稳态动力学分析将用于确定特定氨基酸的一般作用，基于R. etii PC全酶与序列同源，在整体催化机制上。ID和2D NMR实验将用于探测生物素辅基的结构域间运动，并探索生物素和丙酮酸在催化机制中的烯醇化。同位素比质谱分析和内部竞争或远程标记方法将用于测量单个反应步骤的动力学同位素效应，以探测过渡态和相关反应步骤的性质。稳态动力学分析加上同位素效应和NMR实验将使我们能够获得宝贵的洞察到整个催化反应中发生的各个反应步骤。 丙酮酸羧化酶是葡萄糖代谢途径中的重要调控酶。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。为了充分实现PC作为药物靶点的潜力，拟议的研究将侧重于在化学水平上了解PC如何催化肿瘤发生的第一步。

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.