Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase.
Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.
Zeng
Jia
J
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, People's Republic of China.
Li
Ding
D
D000214:Acyl Coenzyme A / Q000737:chemistry; D042964:Acyl-CoA Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D019748:Carbon-Carbon Double Bond Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D064006:Dodecenoyl-CoA Isomerase; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D051381:Rats; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; C034681:octanoyl-coenzyme A; D001224:Aspartic Acid; D018698:Glutamic Acid; D042964:Acyl-CoA Dehydrogenase; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase; C576298:ECI1 protein, human; C119738:ECI2 protein, human