The role of two glutamate residues (E164 and E144) in the active site of enoyl-CoA hydratase has been probed by site-directed mutagenesis. The catalytic activity of the E164Q and E144Q mutants has been determined using 3'-dephosphocrotonyl-CoA. Removal of the 3'-phosphate group reduces the affinity of the substrate for the enzyme, thereby facilitating the determination of K(m) and simplifying the analysis of the enzymes' pH dependence. k(cat) for the hydration of 3'-dephosphocrotonyl-CoA is reduced 7700-fold for the E144Q mutant and 630000-fold for the E164Q mutant, while K(m) is unaffected. These results indicate that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme. Previously, we reported that, in contrast to the wild-type enzyme, the E164Q mutant was unable to exchange the alpha-proton of butyryl-CoA with D(2)O [D'Ordine, R. L., Bahnson, B. J., Tonge, P. J. , and Anderson, V. E. (1994) Biochemistry 33, 14733-14742]. Here we demonstrate that E144Q is also unable to catalyze alpha-proton exchange even though E164, the glutamate that is positioned to abstract the alpha-proton, is intact in the active site. The catalytic function of each residue has been further investigated by exploring the ability of the wild-type and mutant enzymes to eliminate 2-mercaptobenzothiazole from 4-(2-benzothiazole)-4-thiabutanoyl-CoA (BTTB-CoA). As expected, reactivity toward BTTB-CoA is substantially reduced (690-fold) for the E164Q enzyme compared to wild-type. However, E144Q is also less active than wild-type (180-fold) even though elimination of 2-mercaptobenzothiazole (pK(a) 6.8) should require no assistance from an acid catalyst. Clearly, the ability of E164 to function as an acid-base in the active site is affected by mutation of E144 and it is concluded that the two glutamates act in concert to effect catalysis.

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.

Enoyl-CoA hydratase catalyzes the stereospecific hydration of R, -unsaturated acyl-CoA thiolesters. Hydration of trans-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)-hydroxybutyrylCoA with enzyme in D 2 O 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(R)-hydroxybutyryl-CoA formation is 4 × 10 5 -fold slower than the normal hydration reaction, but at least 1.6 × 10 6 -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 × 10 5 . 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.

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.