Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry

Jaswir Basran, Nina Bhanji, Amrik Basran, Daniel Nietlispach, Sharad Mistry, Rolandas Meskys, Nigel S. Scrutton

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    Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s-1. This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme - iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s-1) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s-1) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s-1). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM-1 s-1, respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme - iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent ravin in dimethylglycine oxidase is identified as an αN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.
    Original languageEnglish
    Pages (from-to)4733-4743
    Number of pages10
    Issue number14
    Publication statusPublished - 9 Apr 2002


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