Spectroscopic Insights into the Mechanism of Enoyl-CoA Hydratase

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9604254 Tonge Part 1-Technical The enzyme enoyl-CoA hydratase catalyzes the addition of water across the C-C bond of (,(-unsaturated fatty acid thiol esters. Binding of conjugated subtrate analogs to the enzyme is accompanied by substantial alterations in the electronic structure of the conjugated molecules. The PI describes a series of experiments to investigate the direct relationship between the observed alteration in electronic structure of the substrate analogs and the enzyme's ability to catalyze the hydration of (,(-unsaturated ethylenic bond. The recently described X-ray crystal structure of 4-chlorobenzoyl-CoA dehalogenase, an enzyme that is 28% identical and 50% similar to the hydratase, presents a unique opportunity to dissect the catalytic mechanism of the enzyme. In order to investigate the direct relationship between substrate polarization and enzyme catalysis, residues around the substrate will be systematically modified by site-directed mutagenesis. Combined spectroscopic-reactivity studies will then be performed on each mutant protein to assess the effects of the mutations on catalysis and substrate polarization. These experiments will facilitate the establishment of a direct structure-reactivity correlation for the enzyme-substrate complex and are expected to provide general insight into the role of electrostatics in enzyme catalysis. Part 2- Non Technical Enzymes are proteins that function as biological catalysts, accelerating the rates of chemical reactions by many orders of magnitude. There is intense interest in understanding how enzymes work and in identifying the fundamental basis for enzyme activity. The focus of this proposal is enoyl-CoA hydratase, an enzyme involved in the breakdown of fatty acids in the body. Using site directed mutagenesis, the PI plans to identify and analyze the amino acid residues in the active site of the enzyme that are responsible for the chemical steps of the reaction. A specific goal of this proposal is to quantitatively re late the structure of the substrate in the active site to the rate of the reaction. The structural information will be provided using Raman and NMR spectroscopy. In addition he intends to elucidate the role of the protein's 'electric field' through mutagenesis and monitoring the effect on substrate structure and reactivity. Not only will this information be of great value in understanding the mechanism of enoyl-CoA hydratase, but it will also provide direct insight into the role of electrostatic forces in enzyme catalysis.