STRUCTURE OF HEXADIENOYL-COA BOUND TO ENOYL-COA HYDRATASE DETERMINED BY TRANSFERRED NUCLEAR OVERHAUSER EFFECT MEASUREMENTS - MECHANISTIC PREDICTIONS BASED ON THE X-RAY STRUCTURE OF 4-(CHLOROBENZOYL)-COA DEHALOGENASE

The structure of the substrate analog 2,4-hexadienoyl-coenzyme A (HD-C oA) bound to the enzyme enoyl-CoA hydratase has been determined using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. NOEs between the adenine Hs proton and several pantetheine protons in the b ound form of HD-CoA indicate that the overall structure of the CoA mol ecule is bent, while NOEs between adenine and ribose protons indicate that the conformation about the glycosidic bond is anti. The absence o f long range NOEs along the pantetheine moiety is consistent with this region of the molecule being bound in an extended conformation. In ad dition, NOEs between the vinylic protons indicate that the I-ID moiety is s-trans about C3-C4. The conformation of the CoA portion of bound HD-CoA is strikingly similar to that of the CoA portion of 4-(hydroxyb enzoyl)-CoA bound to the active site of 4-(chlorobenzoyl)-CoA dehaloge nase [Benning, M. M., et al. (1996) Biochemistry 35, 8103--8109]. The structural similarity of the ligands along with the primary sequence h omology validates the modeling of the enoyl-CoA hydratase structure wi th the 4-(chlorobenzoyl)-CoA dehalogenase backbone. The homology model ing allows the prediction that the enoyl-CoA substrates are bound in a n s-cis conformation about C1-C2 and that Glu 144 is present at the ac tive site and can function as a general acid/base.