Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity

Abstract

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.

Functional role of a distal (3′-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction

“…Although the origin of the above apparently contradictory results might be attributed to the difference in the enzyme types, we decided to reinvestigate the effect of the 3h-phosphate on the human liver MCAD (HMCAD)-catalysed reaction with butyryl-CoA and octanoyl-CoA as the enzyme substrates. These substrates were selected for the following reasons : (1) the microscopic pathways of the octanoyl-CoA-and butyryl-CoA-dependent reactions are different [5,6,12], and (2) the rate-limiting step of the enzyme is different with these substrates [13]. By using these substrates we sought to probe the specific role (if any) of the 3h-phosphate group of acyl-CoAs during different steps of the enzyme catalysis.…”

“…The dissociation ' off-rates ' of normal and dephosphoenoylCoAs from the oxidized enzyme site were measured by employing the stopped-flow technique in a sequential-mixing mode [2,6]. This allowed us to minimize the contributions of slow hydration and\or isomerization reactions of enoyl-CoAs.…”

“…In pursuit of delineating the kinetic mechanism of medium-chain acyl-CoA dehydrogenase (MCAD) [1][2][3][4][5][6][7], we became interested in the role(s) of distal (' seemingly useless ') portions of the CoA structure (among acyl-CoA substrates) in the enzyme catalysis. The latter was prompted by our observation that the 3h,5h-ADP fragment of CoA of a chromogenic substrate, 3-indolepropionylCoA (IPCoA), has a crucial role in the enzyme catalysis [8].…”

“…Abbreviations used : CT complex, charge-transfer complex ; ETF, electron-transferring flavoprotein ; FcPF 6 , ferrocenium hexafluorophosphate ; HMCAD, human liver medium-chain acyl-CoA dehydrogenase ; IPCoA, 3-indolepropionyl-CoA ; IPPP, 3-indolepropionyl pantetheine phosphate ; MCAD, medium-chain acyl-CoA dehydrogenase.…”

“…Although the above model was initially proposed for the IPCoA\IPPP-dependent reaction of the enzyme, further experimental data suggested that it was also applicable for the octanoyl-CoA-dependent reaction of the enzyme (as octanoyl-CoA is a physiological substrate of the enzyme) [6].…”