REDUCTIVE HALF-REACTION OF MEDIUM-CHAIN FATTY ACYL-COA DEHYDROGENASE UTILIZING OCTANOYL-COA OCTENOYL-COA AS A PHYSIOLOGICAL SUBSTRATE/PRODUCT PAIR - SIMILARITY IN THE MICROSCOPIC PATHWAYS OF OCTANOYL-COA OXIDATION AND OCTENOYL-COA BINDING

Of the different chain length fatty acyl-CoA substrates, octanoyl-CoA has been known as one of the most efficient (and physiological) substrates for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction of MCAD-FAD with octanoyl-CoA ([MCAD-FAD] << [octanoyl-CoA]), measured via the stopped-flow technique, at 5 degrees C was characterized by a biphasic decrease and increase in absorptions at 450 and 545 nm, respectively. The average values of the fast (1/(tau 1)) and slow (1/(tau 2)) relaxation rate constants, derived from the data at these wavelengths, were found to be 319.7 +/- 33.5 and 28.8 +/- 12.5 s(-1), respectively, and both of these relaxation rate constants remained invariant between 8 and 200 mu M concentrations of octanoyl-CoA. Under identical experimental conditions, we measured time courses for the interaction of MCAD-FAD with octenoyl-CoA ([MCAD-FAD] << [octenoyl-CoA]) by monitoring the absorption changes at 299, 394, and 440 nm. The binding profile was consistent with a biphasic decrease (at 440 nm) and increase (at 299 and 394 nm) in absorbance, with similar magnitudes of fast [1/(tau 1) (average) = 382.3 +/- 39.8 s(-1)] and slow [1/(tau 2) (average) = 14.3 +/- 7.4 s(-1)] relaxation rate constants. The observed relaxation rate constants were, once again, found to be invariant with changes in the octenoyl-CoA concentration from 40 to 150 mu M. In addition, the dissociation rate constant of octenoyl-CoA from the MCAD-FAD-octenoyl-CoA complex (10.3 +/- 0.2 s(-1)) was found to be similar to the overall rate constant (7.1 +/- 0.1 s(-1)) for the release of octanoyl-CoA from the MCAD-FADH(2)-octenoyl-CoA complex (via reversal of the redox reaction). These results attest to a marked similarity in the microscopic pathways of the enzyme-catalyzed oxidation of octanoyl-CoA (i.e., the chemical transformation reaction) and the enzyme-octenoyl-CoA interaction (leading to changes in the electronic structure of FAD). The reductive half-reaction of the enzyme involving octanoyl-CoA vis-a-vis other acyl-CoA substrates led to the following conclusions: (1) Unlike other substrates, the octanoyl-CoA-dependent redox reaction does not limit the overall rate of MCAD catalysis. (2) The higher yield of the reduced enzyme species (80-85%) in the presence of octanoyl-CoA substrate is due to an additional protein isomerization during the reductive half-reaction; this step is absent with other substrates. (3) Due to a slow exchange of octenoyl-CoA by octanoyl-CoA from the MCAD-FADH(2)-octenoyl-CoA complex, the oxidase activity of the enzyme is drastically suppressed. The role of protein conformational changes during the course of ligand binding and/or catalysis is discussed.