Biphasic Kinetic Behavior of E. coli WrbA, an FMNDependent NAD(P)H. Quinone Oxidoreductase

0382149 - ÚVGZ 2013 RIV US eng J - Článek v odborném periodiku
Kishko, Iryna - Harish, B. - Zayats, Vasilina - Řeha, David - Tenner, B. - Beri, D. - Gustavsson, T. - Ettrich, Rüdiger - Carey, J.
Biphasic Kinetic Behavior of E. coli WrbA, an FMNDependent NAD(P)H. Quinone Oxidoreductase.
PLoS ONE. Roč. 7, č. 8 (2012), s. 1-10. ISSN 1932-6203. E-ISSN 1932-6203
Grant CEP: GA ČR GAP207/10/1934
Výzkumný záměr: CEZ:AV0Z60870520
Klíčová slova: Escheria-coli * DT-diaphorase * dehydrogense * mechanism * enzyme * flavoprotein * flavodoxin * reduction
Kód oboru RIV: BO - Biofyzika
Impakt faktor: 3.730, rok: 2012

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. 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.
Trvalý link: http://hdl.handle.net/11104/0212454