Stereospecificity of the reaction catalyzed by enoyl-CoA hydratase

Enoyl-CoA hydratase catalyzes the stereospecific hydration of α,β-unsaturated acyl-CoA thiolesters. Hydration of tras-2-crotonyl-CoA to 3(S)hydroxybutyryl-CoA proceeds via the syn addition of water and thus the pro-2R proton of 3(S)-hydroxybutyryl-CoA is derived from solvent. Incubation of 3(S)-hydroxybutyryl-CoA with enzyme in D₂O results in the slow exchange of the pro-2S proton with solvent deuterium, in addition to the anticipated rapid exchange of the pro-2R proton. Further experiments have shown that the exchange of the pro-2S proton occurs in concert with the formation of the incorrect 3(R)-hydroxybutyryl-CoA enantiomer. The rate of 3(A)-hydroxybutyryl-CoA formation is 4 × 10⁵-fold slower than the normal hydration reaction, but at least 1.6 × 10⁶-fold faster than the non-enzyme-catalyzed reaction. This has allowed us to determine that the absolute stereospecificity for the enzyme-catalyzed reaction is 1 in 4 × 10⁵. The initial formation of 3(R)-hydroxybutyryl-CoA is hypothesized to occur via the incorrect hydration of trans-2-crotonyl-CoA. Once formed, the 3(R)-hydroxybutyryl-CoA dehydrates to give cis-2-crotonyl-CoA. While the equilibrium constant for the hydration of trans-2-crotonyl-CoA to 3(S)-hydroxybutyryl-CoA is 7.5, the equilibrium constant for the hydration of cis-2-crotonyl-CoA to 3(R)-hydroxybutyryl-CoA is estimated to be ∼1000. To validate this reaction scheme, cis-2-crotonyl-CoA has been synthesized and characterized. These studies demonstrate that the enzyme is capable of catalyzing the epimerization of hydroxybutyryl-CoA.