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 microM 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 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.

No abstract

The active site residue, Glu-376, of medium-chain acyl-CoA dehydrogenase (MCAD) has been known to abstract the alpha-proton from acyl-CoA substrates during the course of the reductive half-reaction. The site-specific mutation of Glu-376-->Gln(E376Q) slows down the octanoyl-CoA-dependent reductive half-reaction of the enzyme by about 5 orders of magnitude due to impairment in the proton-transfer step. To test whether the carboxyl group of Glu-376 exclusively serves as the active site base (for abstracting the alpha-proton) during the enzyme catalysis, we undertook a detailed kinetic investigation of the enzyme-ligand interaction and enzyme catalysis, utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair and the wild-type and E376Q mutant enzymes as the catalysts. The transient kinetic data revealed that the E376Q mutation not only impaired the rate of octanoyl-CoA-dependent reduction of the enzyme-bound FAD, but also impaired the association and dissociation rates for the binding of the reaction product, octenoyl-CoA. Besides, the E376Q mutation correspondingly impaired the kinetic profiles for the quenching of the intrinsic protein fluorescence during the course of the above diverse (i.e., "chemistry" versus "physical interaction") processes. A cumulative account of the experimental data led to the suggestion that the carboxyl group of Glu-376 of MCAD is intimately involved in modulating the microscopic environment (protein conformation) of the enzyme's active site during the course of ligand binding and catalysis. Arguments are presented that the electrostatic interactions among Glu-376, FAD, and CoA-ligands are responsible for structuring the enzyme's active site cavity in the ground and transition states of the enzyme during the above physicochemical processes.