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

Biochemistry
Jaswir BasranNigel S Scrutton

Abstract

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 ...Continue Reading

Citations

Jul 4, 2003·Applied and Environmental Microbiology·Ana C Negrete-RaymondLawrence P Wackett
Jun 5, 2004·Protein Expression and Purification·Marcel ZámockýBystrík Polek
Mar 20, 2015·Biopolymers·Jonathan M BurgDewey G McCafferty
Oct 9, 2014·PloS One·Dagmar BruggerClemens K Peterbauer
Mar 26, 2009·Biochemistry·Methinee ProngjitPimchai Chaiyen

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