TY - JOUR
T1 - Oxidation-Reduction of General Acyl-CoA Dehydrogenase by the Butyryl-CoA/Crotonyl-CoA Couple
T2 - A New Investigation of the Rapid Reaction Kinetics
AU - Schopfer, Lawrence M.
AU - Massey, Vincent
AU - Ghisla, Sandro
AU - Thorpe, Colin
PY - 1988/8/1
Y1 - 1988/8/1
N2 - 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/croto-nyl-CoA, oxidized GAD/α,β-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/ crotonyl-CoA (in 50 mM potassium phosphate buffer, pH 7.6 at 4 °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. α,β-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.
AB - 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/croto-nyl-CoA, oxidized GAD/α,β-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/ crotonyl-CoA (in 50 mM potassium phosphate buffer, pH 7.6 at 4 °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. α,β-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.
UR - http://www.scopus.com/inward/record.url?scp=0023809692&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023809692&partnerID=8YFLogxK
U2 - 10.1021/bi00417a059
DO - 10.1021/bi00417a059
M3 - Article
C2 - 3219356
AN - SCOPUS:0023809692
SN - 0006-2960
VL - 27
SP - 6599
EP - 6611
JO - Biochemistry
JF - Biochemistry
IS - 17
ER -