title: 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.
authors.affiliation: Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
authors.forename: L
authors.initials: L
authors.lastname: Falzon
authors.forename: V L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D004583:Electrons; D005486:Flavin Mononucleotide / Q000737:chemistry; D007700:Kinetics; D008744:Methylamines / Q000737:chemistry; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D008744:Methylamines; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C023336:trimethylamine