X-ray crystallography has provided atomic resolution views of substrates and inhibitors bound at enzyme active sites, conveying the impression that the active site functional groups provide a precisely aligned """"""""solvation sphere"""""""" for the ligand. Yet, the resolution of the structures is insufficient to determine the extent that this specific solvation alters the chemical reactivity of the bound molecule. The experiments in this proposal are designed to detect and quantify the electronic strain present in substrates bound to enoyl-CoA hydratase, lactate dehydrogenase and 3-hydroxy-3-methylglutaryl-CoA reductase. The primary methods to be used are spectroscopic, largely resonance Raman and UV, and isotope effects. The proposed studies will increase our understanding of enzyme catalysis and provide the necessary background to design transition state analogs. Crotonase has been proposed to catalyze a concerted syn elimination reaction. Novel H/D/T isotope effect experiments that will confirm the concerted mechanism and establish whether the transition state is characterized by tunneling are proposed. Further we will examine the ground state structure of the substrate and product complexes spectroscopically. Preliminary UV and resonance Raman results indicate there are dramatic alterations in the electronic structure of the bound substrates. Enzymes catalyzing syn eliminations are a growing class of enzyme, including some endonucleases, that are not well characterized mechanistically. Past experimental studies have ignored the function of the carboxamide in dehydrogenase reactions. The extreme stereospecificity of lactate dehydrogenase will be determined with site specific mutants and nucleotide analogs to quantify the importance of the carboxamide in the catalytic mechanism. The induction of strain in the dihydropyridine ring of NADH in the ternary complex will be examined by measuring isotope effects on association. The well defined ion pair and H-bonding interactions of the enzyme with lactate and oxamate will allow us to calibrate the effects of these molecular interactions on isotope effects on association. HMG-CoA reductase is a pharmaceutically important enzyme that has recently been cloned, overexpressed and crystallized. The dithioester of HMG-CoA is a potent inhibitor of this enzyme, which additionally induces substrate inhibition by NAD(P)H. We plan to pursue stereochemical, spectroscopic and kinetic studies of this enzyme to delineate the importance of the thiocarbonyl polarization to the unusual inhibition.
Showing the most recent 10 out of 34 publications
