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.
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