ENOYL-COA HYDRATASE AND ISOMERASE FORM A SUPERFAMILY WITH A COMMON ACTIVE-SITE GLUTAMATE RESIDUE

Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily. Their substrates and reaction mechanisms are similar [Muller-Newen, G. and Stoffel, W. (1993) Biochemistry 32, 11405-11412]. Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities. We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction. We functionally expressed rat liver mECH wild-type and [E164Q] mutant enzymes in Escherichia coli. Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the k(cat) value more than 100000-fold, whereas the K-m value is only moderately affected. We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity. Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.