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 bi-substrate 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.
Highlights
The WrbA protein from E. coli [1] has been identified as the founding member of a family of novel flavoproteins conserved from bacteria to higher plants [2] whose exact physiological role is still unknown [3]
The results presented here are consistent with a kinetic mechanism of the ping-pong type for WrbA, though with at least two forms of the enzyme present as evidenced by the product inhibition and NMR results
The analysis presented here suggests that the nonphysiological location of the nicotinamide dinucleotide in the NADH-soaked WrbA crystal [14] represents a stage of dissociation of the oxidized product NAD
Summary
The WrbA protein from E. coli [1] has been identified as the founding member of a family of novel flavoproteins conserved from bacteria to higher plants [2] whose exact physiological role is still unknown [3]. The crystal structures of WrbAs [10] reveal cavernous active sites with a chamber above the flavin isoalloxazine ring system that appears to be big enough to accommodate either NADH as electron donor or benzoquinone (BQ; BQH2 is reduced BQ) as electron acceptor, but not both substrates simultaneously. This structural evidence suggests that the kinetic mechanism of WrbA is of the ping-pong type, as has been shown for NQO1 [11]. The present analysis was undertaken to evaluate this suggestion and to compare the steady-state kinetic properties of WrbA with the peculiar properties reported previously for NQO1
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