Microscopic pathway for the medium chain fatty acyl CoA dehydrogenase catalyzed oxidative half-reaction: Changes in the electronic structures of flavin and CoA derivatives during catalysis

Abstract

In a previous communication, we demonstrated that the medium-chain fatty acyl CoA dehydrogenase (MCAD) catalyzed conversion of 3-indolepropionyl CoA (IPCoA) to trans-3-indoleacryloyl CoA (IACoA) proceeds via the formation of an intermediary species X that possesses the electronic properties of reduced flavin and highly conjugated CoA product. Since the steady-state turnover of the enzyme-catalyzed dehydrogenation reaction precisely matches with the rate of formation of X [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013], the latter species appeared to be the likely site for the transfer of electrons to external electron acceptors (e.g., ferricenium hexafluorophosphate, FcPF6). To probe the microscopic pathway for the oxidative half-reaction, we employed a sequential mixing stopped-flow technique utilizing IPCoA as the enzyme substrate and FcPF6 as the electron acceptor. The time-dependent changes in absorption at 450, 415, and 367 nm were measured upon mixing FcPF6 with previously mixed and aged solutions of MCAD-FAD+IPCoA in the stopped-flow syringes. The kinetic traces show an increase (1/tau 1) followed by a decrease (1/tau 2) in absorption at 450 and 415 nm, and a lag (corresponding to the time regime of 1 u 1) followed by an increase in absorption (1/tau 2) at 367 nm. The relaxation rate constants (1/tau's) thus measured remain unaffected, with variations in the aging time; however, the amplitudes of these phases increase up to the aging time of 5 s, after which the amplitudes attain maxima. For an aging time of 5 s, 1/tau 1 and 1/tau 2 show a linear and a hyperbolic dependence on the FcPF6 concentration, respectively. These, coupled with the complementary studies involving butyryl CoA as a nonchromophoric substrate for this enzyme, lead us to propose the following sequence of events during the MCAD-catalyzed oxidative half-reaction: (1) The enzyme-catalyzed oxidative half-reaction proceeds via the formation of a collision complex between X and FcPF6 during the fast (1/tau 1) relaxation phase. (2) The reduced flavin moiety of X is oxidized via (rapid) transfer of electrons to FcPF6 within the collision complex, without formation of a detectable (metastable) flavin semiquinone intermediate. (3) The transfer of electrons is accompanied by changes in the electronic structures of both the flavin and IACoA moieties within the enzyme-IACoA complex. The electronic structure of this newly formed complex is exactly the same as that formed upon isomerization of the MCAD-FAD-IACoA complex [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575].(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…The transient kinetic experiments were performed with an Applied Photophysics SX-17MV sequential-mixing stopped-flow system (optical path length 10 mm, dead time 1.3-1.5 ms) in the single or sequential-mixing modes [2,5,14]. The stopped-flow kinetic traces were analysed by the data analysis package provided by Applied Photophysics.…”

“…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.…”

“…We investigated the transient kinetics for the oxidative halfreaction of the enzyme with both normal and 3h-dephospho forms of octanoyl-CoA and butyryl-CoA as substrates and FcPF ' as electron acceptor via the sequential-mixing stoppedflow method [2]. During these experiments the substrate-reduced enzyme was freshly generated by mixing HMCAD-FAD with either of the above substrates via the first two stopped-flow syringes.…”

“…17.1 kJ\mol. (2) This deletion of the 3h,5h-ADP fragment of IPCoA destabilizes the intermediary CT complex. As a result, the CT complex is not readily detectable during the IPPPdependent reductive half-reaction of the enzyme.…”

“…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].…”