Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups.
The trimethylamine dehydrogenase of bacterium W3A1 is reduced with the formation of a triplet state in which two electrons, derived from the substrate, are distributed between the [4Fe-4S] cluster and 6-S-cysteinyl-FMN semiquinone. In titration experiments at pH 8.5 about 1.0 mol of dimethylamine or 0.5 mol of trimethylamine per mol of the enzyme is required to titrate the enzyme to an endpoint. At pH values less than 8.0, however, an excess of trimethylamine is required to obtain maximal yield of the g = 4 e.p.r. signal, characteristic of the triplet state, or maximal absorbance at 365 nm which indicates formation of the flavin semiquinone. The binding of 0.86 mol of trimethylamine per mol of the enzyme could be detected by a gel chromatographic method. When the enzyme is titrated with dithionite in the presence of tetramethylammonium chloride, an endpoint is reached after the uptake of two electrons which give rise to the triplet state, whereas three electrons are consumed in the absence of tetramethylammonium chloride to reduce the enzyme completely. The enzyme is inhibited noncompetitively by tetramethylammonium chloride and the slopes of double reciprocal plots are a concave upwards function of inhibitor concentration. The data indicate the presence of a binding site for the substrate and other amines on the reduced enzyme which enhances the proportion of enzyme in the triplet state.
D001419:Bacteria / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008744:Methylamines / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013053:Spectrophotometry
D046911:Macromolecular Substances; D008744:Methylamines; D000644:Quaternary Ammonium Compounds; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C027917:tetramethylammonium; C023336:trimethylamine