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 [Müller-Newen, G. & Stoffel, W. (1993) Biochemistry 32, 11,405-11,412]. 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 kcat value more than 100,000-fold, whereas the Km 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.
Müller-Newen
G
G
Institute of Biochemistry, Medical Faculty, University of Cologne, Germany.
Janssen
U
U
Stoffel
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D018698:Glutamic Acid; D004746:Enoyl-CoA Hydratase; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase