Dissecting the mechanism of enoyl-CoA hydratase

Enoyl-CoA hydratase (ECH) is the second enzyme in the fatty acid β-oxidation pathway that occurs in mammalian cells in both mitochondria and peroxisomes. It catalyzes the syn addition of water across the C=C bond of α,β unsaturated fatty acid thiolesters. In my research, Raman, FTIR and NMR spectroscopy are used in concert with site-directed mutagenesis and kinetics to investigate the mechanism of the reaction catalyzed by ECH. These methods probe the alterations in the electronic structure of both the substrates and substrate analogs that occur upon binding to the enzyme. Site-directed mutagenesis is then used to modify the protein and spectroscopic and kinetic characterization of the mutant enzymes enable structure-reactivity correlations to be generated.; The roles of two glutamate residues (E164 and E144) in the active site of enoyl-CoA hydratase have been probed by site-directed mutagenesis and kinetic pH dependence. Replacing either glutamate with glutamine or aspartate results in significant decrease in the activity with K_m unaffected, indicating that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme and that the precise positioning and orientation of the catalytic residues in the active site is critical for efficient substrate hydration. We determined the kinetic pH dependence for wild type and E144D, E16413, E144Q, E164Q and E144/164Q mutants. The bell shaped pH dependence of k_cat for wild-type and E164D shows two ionizable groups in the active site, whereas E164Q and E144Q only show one ionizable group. The double mutant E164/144Q doesn't show obvious pH dependence. These results provide strong evidence for E144 and E164 to be catalytic acids and bases. The fact that the pH dependence of the k_cat for both E164Q and E144Q mutants shows an ascending curve suggests that the active form of the enzyme is one in which one of the glutamates (wild-type) or the one remaining glutamate (E144Q, E164Q must be ionized for the reaction to occur.; The peptidic NH groups of G141 and A98 comprise the oxyanion hole for stabilizing negative charge accumulation on the thioester carbonyl group. The significant decrease in the k_cat of A98P and G141P indicates the H-bond is crucial for the catalysis. Raman difference spectra of hexadienoyl-CoA(HD-CoA) bound to wild-type ECH shows large wavenumber shifts and intensity increases in the C=C stretching modes of HD-CoA relative to the free ligand, resulting from the substrate polarization in the active site. For G141P, this polarization is completely lost, demonstrating the importance of the hydrogen bond to the carbonyl oxygen in substrate polarization. (Abstract shortened by UMI.)...