Detailed mechanistic aspects of carbohydrate metabolism are being studied. Emphasis is being placed on several enzymes which are involved in key steps in the synthesis and in the degradation of carbohydrate-energy storage and energy utilization. These enzymes are important in metabolic function in the liver, muscle and brain and improper regulation leads to complications in diabetes and apparently in Infant Death Syndrome. The primary enzymes under scrutiny are phosphoenolpyruvate carboxykinase, enolase and pyruvate kinase. The multiple roles which the activating cations play in the chemical mechanism and in the regulation of catalytic activity are being elucidated by kinetic and physical studies. The application of water proton and substrate (1H, 13C, 19F, 31P) nuclear relaxation rate techniques are utilized to determine the location of the cations, their functions and their effects on ligand bindings. The locations of the metal ions with respect to each other will be measured by EPR and PRR methods. The environments of these cations will be determined. From nuclear relaxation rate studies the structures of the substrates and the regulators at the cation site and their exchange rates will be determined. Novel applications of protein and of metal magnetic resonance studied will be attempted to describe the nature of ligand-protein interactions, activation of substrates and activation of the enzyme. Synergistic effects of ligand binding will be described and quantitated. These ligand effects will be utilized to design activators or inhibitors of key enzymes in carbohydrate metabolism. Such an approach to enzyme activation/inhibition could prove fruitful for other enzyme systems. Comparative enzymology will be studied to search for enzyme homology. Such an approach to enzymology may yield additional insight into mechanistic details of this group of enzymes.
