Oxidation-reduction of general acyl-CoA dehydrogenase by the butyryl-CoA/crotonyl-CoA couple. A new investigation of the rapid reaction kinetics.
Pig kidney general acyl-CoA dehydrogenase (GAD) can be reduced by butyryl-CoA to form reduced enzyme and crotonyl-CoA. This reaction is reversible. Stopped-flow, kinetic investigations on GAD have been made, using the following reaction pairs: oxidized GAD/butyryl-CoA, oxidized GAD/crotonyl-CoA, oxidized GAD/alpha,beta-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/crotonyl-CoA (in 50 mM potassium phosphate buffer, pH 7.6 at 4 degrees C). Reduction of GAD by butyryl-CoA is triphasic. The slowest phase is 100-fold slower than the preceding phase and appears to represent a secondary process not directly related to the primary reduction events. The first two fast phases are responsible for reduction of GAD. Reduction proceeds via a reduced enzyme/crotonyl-CoA charge-transfer complex. alpha, beta-Dideuteriobutyryl-CoA elicits a major deuterium isotope effect (15-fold) on the reduction reaction. Oxidation of GAD by crotonyl-CoA is biphasic. Oxidation proceeds via the same reduced enzyme/crotonyl-CoA charge-transfer complex seen during reduction. The oxidation reaction ends in a mixture composed largely of oxidized GAD species. From the data, we constructed a mechanism for the reduction/oxidation of GAD by butyryl-CoA/crotonyl-CoA. This mechanism was then used to simulate all of the observed kinetic time course data, using a single set of kinetic parameters. A close correspondence between the observed and simulated data was obtained.
Schopfer
L M
LM
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Massey
V
V
Ghisla
S
S
Thorpe
C
C
D000214:Acyl Coenzyme A / Q000378:metabolism*; D044944:Acyl-CoA Dehydrogenases / Q000378:metabolism*; D000818:Animals; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine
D000214:Acyl Coenzyme A; C024343:butyryl-coenzyme A; C010701:crotonyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases