Pyruvate carboxylase is a member of the family of biotin-dependent enzymes which play key roles in intermediary metabolism. Pyruvate carboxylase deficiencies mostly occur in neonates or infants and result in severe psychomotor retardation leading to early death. Over expression of hepatic pyruvate carboxylase is associated with type II diabetes and obesity. The purpose of this project is to determine the first three dimensional structure of pyruvate carboxylase as an example of an intact biotin-dependent holoenzyme, and to perform a detailed structure-function analysis on this enzyme. We shall: (1) Perform crystallization trials on pyruvate carboxylases from a number of sources with the aim of determining the 3-D structure of the enzyme by X-ray crystallographic analysis. (2) Determine the binding site of the physiological, allosteric activator, acetyl CoA by co-crystallization with a non-hydrolysable analogue and by affinity labeling. (3) Determine the regions of polypeptide sequence involved in the action of acetyl CoA by producing chimeric constructs of pyruvate carboxylases from species which show different degrees of response to acetyl CoA. By performing site-directed mutagenesis on specific amino acid residues in the enzyme and kinetic analyses of the wild-type and mutant enzymes using a combination of 2H, 13C and 18O kinetic isotope effect experiments, and steady-state and pre-steady state kinetic analysis to pinpoint the precise roles of these residues in the catalytic mechanism of the enzyme we shall elucidate (4) The catalytic mechanism of the biotin carboxylation reaction common to the majority of biotin-dependent carboxylases in the context of a holoenzyme. (5) The catalytic mechanism of the carboxylation of pyruvate by carboxybiotin and movement of carboxybiotin between the sites of the partial reactions in the enzyme active site. (6) The action of acetyl CoA at the level of the catalytic mechanism. (7) We shall synthesize carboxyphosphate and demonstrate its ability to act as an intermediate in the pyruvate carboxylase reaction.
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