Enoyl-Coenzyme A Hydratase-Catalyzed Exchange of the α-Protons of Coenzyme A Thiol Esters: A Model for an Enolized Intermediate in the Enzyme-Catalyzed Elimination?

3-Quinuclidinone catalyzes the exchange of the α-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10^-6 M^-1 s^-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10² M^-1 s^-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pK_a of 8.8 and below a pK_a of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10⁵ as no incorporation of ²H into the α-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the α-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the α-protons becomes nonstereospecific when the acidity of the α-protons is enhanced. While α-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.