Biphasic kinetic behavior of FMN-dependent NAD(P)H:quinone oxidoreductase WrbA from E. coli: A molecular understanding

David Reha¹, Vasilina Zayats¹ , Balasubramain Harish² , Iryna Kishko¹ , Jannette Carey² and Rüdiger Ettrich¹

¹Institute of Nanobiology and Structural Biology GCRC, Academy of Sciences of the Czech Republic, Nové Hrady, e-mail: ettrich@nh.cas.cz

²Department of Chemistry, Princeton University, Princeton, New Jersey, USA

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. 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. 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. A novel approach of polarized QM/MM molecular docking of substrates to the dimar and tetramer demonstrates different binding affinities for the dimer and tetramer and supports the hypothesis that coupling of substrate binding to tetramer assembly may underlie the two-plateau kinetics.

Support from the Czech Science Foundation, no P207-10-1934, is acknowledged.