Stereoselectivity of enoyl-CoA hydratase results from preferential activation of one of two bound substrate conformers.

Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)- and 3(R)-hydroxybutyryl-CoA with a stereoselectivity (3(S)/3(R)) of 400,000 to 1. Importantly, Raman spectroscopy reveals that both the s-cis and s-trans conformers of the substrate analog hexadienoyl-CoA are bound to the enzyme, but that only the s-cis conformer is polarized. This selective polarization is an example of ground state strain, indicating the existence of catalytically relevant ground state destabilization arising from the selective complementarity of the enzyme toward the transition state rather than the ground state. Consequently, the stereoselectivity of the enzyme-catalyzed reaction results from the selective activation of one of two bound substrate conformers rather than from selective binding of a single conformer. These findings have important implications for inhibitor design and the role of ground state interactions in enzyme catalysis.

Effect of mutagenesis on the stereochemistry of enoyl-CoA hydratase.

Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)-HB-CoA, 3(S)-hydroxybutyryl-CoA with a stereospecificity (k(S)/k(R)) of 400000 to 1 [Wu, W. J., Feng, Y., He, X., Hofstein, H. S., Raleigh, D. P., and Tonge, P. J. (2000) J. Am. Chem. Soc. 122, 3987-3994]. Replacement of E164, one of the catalytic glutamates in the active site, with either aspartate or glutamine reduces the rate of formation of the 3(S) product enantiomer (k(S)) without affecting the rate of formation of the 3(R) product (k(R)). Consequently, k(S)/k(R) is 1000 and 0.33 for E164D and E164Q, respectively. In contrast, mutagenesis of E144, the second catalytic glutamate, reduces the rate of formation of both product enantiomers. Thus, only E144 is required for the formation of 3(R)-HB-CoA, 3(R)-hydroxybutyryl-CoA. Modeling studies together with analysis of alpha-proton exchange rates and experiments with crotonyl-oxyCoA, a substrate analogue in which the alpha-proton acidity has been reduced 10000-fold, support a mechanism of 3(R)-hydroxybutyryl-CoA formation that involves the E144-catalyzed stepwise addition of water to crotonyl-CoA which is bound in an s-trans conformation in the active site. Finally, we also demonstrate that hydrogen bonds in the oxyanion hole, provided by the backbone amide groups of G141 and A98, are important for the formation of both product enantiomers.