CRYSTAL-STRUCTURE OF ENOYL-COENZYME-A (COA) HYDRATASE AT 2.5 ANGSTROMRESOLUTION - A SPIRAL FOLD DEFINES THE COA-BINDING POCKET

The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA ) hydratase complexed with the potent inhibitor acetoacetyl-CoA has be en refined at 2.5 Angstrom resolution. This enzyme catalyses the rever sible addition of water to alpha beta-unsaturated enoyl-CoA thioesters , with nearly diffusion-controlled reaction rates for the best substra tes, Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex, The hexamer is a dimer of trimers . The monomer is folded into a right-handed spiral of four turns, foll owed by two small domains which are involved in trimerization, Each tu rn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereoc hemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It conf irms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importan ce of Glu144 as the catalytic base for the activation of a water molec ule in the hydratase reaction. The comparison of an un-liganded and a liganded active site within the same crystal form shows a water molecu le in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water durin g catalysis.