Relative Timing of Hydrogen and Proton Transfers in the Reaction of Flavin Oxidation Catalyzed by Choline Oxidase

Gannavaram, Swathi; Gadda, Giovanni

doi:10.1021/bi3016235

Cited by 22 publications

(55 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…H/D exchange at N5-FMN red was documented but did not explain the small KIE. A similar primary KIE of only 2 was observed for complex I and was attributed to hydride transfer’s being only partially rate-determining (Birrell and Hirst, 2013) and a primary KIE of ~1.5 was also observed in the flavin-oxidizing half-reaction of choline oxidase (Gannavaram and Gadda, 2013). More detailed studies are required to establish whether the chemical step is solely responsible for our primary KIE, however this interpretation is strengthened by the similarity of the KIEs on k cat and on k cat /K M .…”

Section: Discussionsupporting

confidence: 54%

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

et al. 2017

View full text Add to dashboard Cite

Summary Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire.

show abstract

Section: Discussionsupporting

confidence: 54%

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

et al. 2017

View full text Add to dashboard Cite

show abstract

“…29 In contrast, the flavooxidase choline oxidase proved to transfer both the H and H + to O 2 in a concerted manner, in the same chemical step, as arisen from the studies of multiple isotope effects on k cat /K ox . 36…”

Section: Agingmentioning

confidence: 99%

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase

et al. 2018

Self Cite

View full text Add to dashboard Cite

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α-H]- p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.

show abstract

“…To the best of our knowledge, the mechanism of the oxidative half-reaction was investigated in detail in three oxidases, namely pyranose 2-oxidase, 30,31 choline oxidase 34 and arylalcohol oxidase. 35 It was found that their mechanisms con- cerning the sequence and relative energetics of the elementary steps are different.…”

Section: Introductionmentioning

confidence: 99%

A detailed mechanism of the oxidative half-reaction ofd-amino acid oxidase: another route for flavin oxidation

Kiss

Ferenczy

2019

Org. Biomol. Chem.

View full text Add to dashboard Cite

show abstract

Relative Timing of Hydrogen and Proton Transfers in the Reaction of Flavin Oxidation Catalyzed by Choline Oxidase

Cited by 22 publications

References 38 publications

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase

A detailed mechanism of the oxidative half-reaction ofd-amino acid oxidase: another route for flavin oxidation

Contact Info

Product

Resources

About