Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H: Quinone Oxidoreductase
(2012) In PLoS ONE 7(8).- Abstract
- The E. coli protein WrbA is an FMN-dependent NAD(P)H: quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive... (More)
- The E. coli protein WrbA is an FMN-dependent NAD(P)H: quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bisubstrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H: quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots. (Less)
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https://lup.lub.lu.se/record/3147120
- author
- Kishko, Iryna ; Harish, Balasubramanian ; Zayats, Vasilina ; Reha, David ; Tenner, Brian ; Beri, Dhananjay ; Gustavsson, Tobias LU ; Ettrich, Ruediger and Carey, Jannette
- organization
- publishing date
- 2012
- type
- Contribution to journal
- publication status
- published
- subject
- in
- PLoS ONE
- volume
- 7
- issue
- 8
- publisher
- Public Library of Science (PLoS)
- external identifiers
-
- wos:000308206000058
- scopus:84865607387
- pmid:22952804
- ISSN
- 1932-6203
- DOI
- 10.1371/journal.pone.0043902
- language
- English
- LU publication?
- yes
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- a8363da1-b0e7-42bb-beea-8969e60d13e0 (old id 3147120)
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@article{a8363da1-b0e7-42bb-beea-8969e60d13e0,
abstract = {{The E. coli protein WrbA is an FMN-dependent NAD(P)H: quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bisubstrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H: quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.}},
author = {{Kishko, Iryna and Harish, Balasubramanian and Zayats, Vasilina and Reha, David and Tenner, Brian and Beri, Dhananjay and Gustavsson, Tobias and Ettrich, Ruediger and Carey, Jannette}},
issn = {{1932-6203}},
language = {{eng}},
number = {{8}},
publisher = {{Public Library of Science (PLoS)}},
series = {{PLoS ONE}},
title = {{Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H: Quinone Oxidoreductase}},
url = {{http://dx.doi.org/10.1371/journal.pone.0043902}},
doi = {{10.1371/journal.pone.0043902}},
volume = {{7}},
year = {{2012}},
}