Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry.
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) x 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 flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.
Basran
Jaswir
J
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
Bhanji
Nina
N
Basran
Amrik
A
Nietlispach
Daniel
D
Mistry
Sharad
S
Meskys
Rolandas
R
Scrutton
Nigel S
NS
D001173:Arthrobacter / Q000201:enzymology*; D005420:Flavoproteins / Q000378:metabolism; D005557:Formaldehyde / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
D005420:Flavoproteins; D005557:Formaldehyde; C025138:dimethylglycine; D006861:Hydrogen Peroxide; D000587:Oxidoreductases Acting on CH-NH Group Donors; C431689:dimethylglycine oxidase; D010100:Oxygen; D012521:Sarcosine