PMID:7883013
| Citation |
Müller-Newen, G, Janssen, U and Stoffel, W (1995) Enoyl-CoA hydratase and isomerase form a superfamily with a common active-site glutamate residue. Eur. J. Biochem. 228:68-73 |
|---|---|
| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Carbon-Carbon Double Bond Isomerases; Enoyl-CoA Hydratase/chemistry; Enoyl-CoA Hydratase/isolation & purification; Enoyl-CoA Hydratase/metabolism; Escherichia coli/genetics; Glutamic Acid/metabolism; Isomerases/chemistry; Isomerases/isolation & purification; Isomerases/metabolism; Molecular Sequence Data; Rats; Recombinant Proteins/metabolism; Structure-Activity Relationship |
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