Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.
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.
Peterson
K L
KL
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Sergienko
E E
EE
Wu
Y
Y
Kumar
N R
NR
Strauss
A W
AW
Oleson
A E
AE
Muhonen
W W
WW
Shabb
J B
JB
Srivastava
D K
DK
D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016208:Databases, Factual; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine
D000214:Acyl Coenzyme A; D005227:Fatty Acids; D011994:Recombinant Proteins; C094936:octenoyl-coenzyme A; C034681:octanoyl-coenzyme A; C010667:acetoacetyl CoA; D005182:Flavin-Adenine Dinucleotide; C058947:2-octynoyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase