The objectives of the project are to gain a better understanding of the mechanisms of enzymatic catalysis and of the means by which inorganic cations contribute to the catalytic processes. Pyruvate kinase and enolase, enzymes from the glycolytic pathway, are of central importance to carbohydrate metabolism in all living organisms. Both enzymes require specific activation by two equivalents of divalent cation, and pyruvate kinase requires activation by potassium as well. Each enzyme exhibits one or more instances of general acid-base catalysis during the catalytic cycle. The roles of magnesium and other divalent cations in enzymatic catalysis are poorly understood. General acid-base catalysis is a topic of current interest in enzymology. Studies of enolase and pyruvate kinase hold promise for revealing fundamental concepts in these areas.
The specific aims of the project focus on key aspects of activation by metal ions and of general acid-base catalysis that have been revealed in previous studies. X-ray crystallography, site-directed mutagenesis, spectroscopic, and kinetic methods are proposed to approach the specific aims which include experiments to: 1) determine the pKa's of the general acid-base catalysts in the active site of enolase; 2) probe the basis for metal specificity in the steps of enolase catalysis; 3) correlate structure and function of wild type and site specific mutant enolases; 4) search for the group or groups responsible for general acid-base catalysis and for a potential proton relay network in the active site of pyruvate kinase ; 5) probe the structural outcome of a Glu to Lys mutation in pyruvate kinase that eliminates the requirement for activation by potassium and probe the structural changes induced by an allosteric effector.
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