Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules

Biochemistry. 1995 Nov 14;34(45):14942-53. doi: 10.1021/bi00045a039.


We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acyl Coenzyme A / metabolism
  • Acyl Coenzyme A / pharmacology
  • Acyl-CoA Dehydrogenase
  • Acyl-CoA Dehydrogenases / antagonists & inhibitors
  • Acyl-CoA Dehydrogenases / chemistry
  • Acyl-CoA Dehydrogenases / isolation & purification
  • Acyl-CoA Dehydrogenases / metabolism*
  • Animals
  • Databases, Factual
  • Fatty Acids / chemistry
  • Fatty Acids / metabolism
  • Flavin-Adenine Dinucleotide / metabolism
  • Humans
  • Kidney / enzymology
  • Kinetics
  • Liver / enzymology
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Swine


  • Acyl Coenzyme A
  • Fatty Acids
  • Recombinant Proteins
  • octenoyl-coenzyme A
  • octanoyl-coenzyme A
  • acetoacetyl CoA
  • Flavin-Adenine Dinucleotide
  • 2-octynoyl-coenzyme A
  • Acyl-CoA Dehydrogenases
  • Acyl-CoA Dehydrogenase