Kinetic Model for the Regulation by Substrate of Intramolecular Electron Transfer in Trimethylamine Dehydrogenase

Abstract

The reaction of trimethylamine dehydrogenase (TMADH) with trimethylamine has been studied by rapid-scanning stopped-flow spectroscopy and steady-state kinetics. The covalently bound 6-S-cysteinylflavin mononucleotide (FMN) cofactor is initially reduced by substrate and exhibits a limiting first order rate constant of 230 s(-1) at pH 7.5 and 30 degrees C. One electron is then transferred intramolecularly from the reduced FMNH2 to the oxidized [4Fe-4S]2+ center. This reaction is biphasic, and the extent of the reaction which corresponds to the faster and slower rates is dependent upon the concentration of trimethylamine. The limiting first order rate constants are 160 and 4 s(-1). At low substrate concentrations, the faster rate is dominant, and at high substrate concentrations, the slower rate is dominant. These results are used to develop a model for the reductive half-reaction of TMADH in which two molecules of substrate bind to TMADH. One binds at the active site of oxidized TMADH and is converted to products. A second molecule binds but is not converted to products and influences the rate of intramolecular electron transfer. Analysis of the transient kinetic data yielded apparent dissociation constants for trimethylamine of 36 and 148 mu M, respectively, for binding to the catalytic and noncatalytic sites. Steady-state kinetic studies indicated substrate inhibition which was best described by a model in which binding of a second molecule of trimethylamine causes a 10-fold reduction in k(cat) from 11 to 1.1 s(-1). This suggests that, at high substrate concentrations, the rate of the intramolecular electron transfer reaction has become sufficiently slow to be at least partially rate-limiting for the steady-state reaction. These kinetic data are interpreted in the context of the known crystal structure of TMADH. The mechanistic implications regarding long range electron transfer and possible physiologic significance of these findings are discussed.

The Reaction of Trimethylamine Dehydrogenase with Trimethylamine

“…The magnitude of these rate constants are generally consistent with the half-life for the fast phase at pH 7.7 of 1.5-2 ms reported by Beinert and co-workers (10) when the higher temperature utilized in this earlier study is taken into account. In a more recent study, however, the kinetic properties for the reductive half-reaction of TMADH have been reported that differ significantly from those reported here (19), with limiting rate constants for the fast phase of 230 s Ϫ1 at pH 7.5, 30°C (significantly smaller than those reported here). In addition, the present results indicate that the slower phases of the reaction exhibit excess substrate inhibition, while this more recent work reported hyperbolic substrate concentration dependence for both the intermediate and slow phases.…”

“…An analogous mechanism has also been shown to account for the excess substrate inhibition observed with xanthine oxidase (31). We emphasize that this mechanism for excess substrate inhibition in the steady-state is a necessary consequence of the known properties of TMADH, and is distinct from a model in which a second, inhibitory, substrate-binding site is present in the enzyme, as has been suggested (19). We note that the x-ray crystal structure of TMADH in complex with the substrate analog TMAC gives no indication of the presence of a second substrate-binding site (24,32).…”

“…EPR Studies-Formation of the spin-interacting state in TMADH is associated primarily with the slow phase of the reductive half-reaction (13)(14)(15), and has previously been interpreted as involving a redistribution of reducing equivalents within two-electron reduced enzyme as product dissociates and a second equivalent of substrate binds (18). More recently, however, it has been ascribed to the binding of a second equivalent of substrate to a discrete inhibitory site on the enzyme (19). In order to investigate whether a second substrate-binding site exists on TMADH whose occupancy is required for formation of the spin-interacting state, the following experiment has been performed, taking advantage of the well known observation that binding of the substrate analog TMAC to partially reduced TMADH elicits the spin-interacting state (24).…”

“…On the basis of the kinetic studies with diethylmethylamine, an overall reductive half-reaction mechanism for TMADH has been proposed (15,18): the fast phase represents the two electron reduction of the flavin cofactor (oxidation of the substrate) with simultaneous formation of a covalent substrate-flavin intermediate; the intermediate phase reflects intramolecular electron transfer from reduced flavin to the 4Fe/4S center, generating flavin semiquinone and reduced 4Fe/4S center with intrinsic rapid electron transfer rate limited by the decay of the covalent adduct (15); and the slow phase involves dissociation of product and binding of a second substrate molecule, which perturbs the electron distribution in the partially reduced enzyme and facilitates formation of the spin-interacting state. Most recently, the reaction with trimethylamine has been re-examined with a considerably slower rate constant for the first phase of the reaction being reported despite the fact that the experiment was performed at 30°C (19). In addition, a kinetic mechanism involving an additional inhibitory substrate-binding site was proposed, although there is no independent evidence for the existence of such an additional substrate binding site.…”

The Role of Tyr-169 of Trimethylamine Dehydrogenase in Substrate Oxidation and Magnetic Interaction between FMN Cofactor and the 4Fe/4S Center

“…Steady-state Kinetic Analyses-As reported previously, the steady-state kinetics of wild-type TMADH exhibit excess substrate inhibition (18,30,31). By contrast, Y169F TMADH exhibits well behaved steady-state behavior: no evidence for substrate inhibition was seen even at very high substrate concentrations (up to 45 mM; Fig.…”