Role of glutamate 144 and glutamate 164 in the catalytic mechanism of enoyl-CoA hydratase

The role of two glutamate residues (E164 and E144) in the active site of en oyl-CoA hydratase has been probed by site-directed mutagenesis. The catalyt ic activity of the E164Q and E144Q-mutants has been determined using 3'-dep hosphocrotonyl-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 E144 Q mutant and 630000-fold for the E164Q mutant, while K-m is unaffected. The se 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 exchang e the or-proton of butyryl-CoA with D2O [D'Ordine; R. L., Bahnson, B. J., T onge, 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-p roton, is intact in the active site. The catalytic function of each residue has been further investigated by exploring the ability of the wild-type an d mutant enzymes to eliminate 2-mercaptobenzothiazole from 4-(2-benzothiazo le)-4-thiabutanoyl-CoA (BTTB-CoA). As expected, reactivity toward BTTB-CoA is substantially reduced (690-fold) for the E164Q enzyme compared to wild-t ype. However, E144Q is also less active than wild-type (180-fold) even thou gh elimination of 2-mercaptobenzothiazole (pK(a) 6.8)should require no assi stance from an acid catalyst. Clearly, the ability of E164 to function as a n acid-base in the active site is affected by mutation of E144 and it is co ncluded that the two glutamates act in concert to effect catalysis.