Kinetic Mechanism of Quinone Oxidoreductase 2 and Its Inhibition by the Antimalarial Quinolines

Abstract

Quinone oxidoreductase 2 (QR2) purified from human red blood cells was recently shown to be a potential target of the quinoline antimalarial compounds [Graves et al., (2002) Mol. Pharmacol. 62, 1364]. QR2 catalyzes the two-electron reduction of menadione via the oxidation of N-alkylated or N-ribosylated nicotinamides. To investigate the mechanism and consequences of inhibition of QR2 by the quinolines further, we have used steady-state and transient-state kinetics to define the mechanism of QR2. Importantly, we have shown that QR2 when isolated from an overproducing strain of E. coli is kinetically equivalent to the enzyme from the native human red blood cell source. We observe ping-pong kinetics consistent with one substrate/inhibitor binding site that shows selectivity for the oxidation state of the FAD cofactor, suggesting that selective inhibition of the liver versus red blood cell forms of malaria may be possible. The reductant N-methyldihydronicotinamide and the inhibitor primaquine bind exclusively to the oxidized enzyme. In contrast, the inhibitors quinacrine and chloroquine bind exclusively to the reduced enzyme. The quinone substrate menadione, on the other hand, binds nonspecifically to both forms of the enzyme. Single-turnover kinetics of the reductive half-reaction are chemically and kinetically competent and confirm the inhibitor selectivity seen in the steady-state experiments. Our studies shed light on the possible in vivo potency of the quinolines and provide a foundation for future studies aimed at creating more potent QR2 inhibitors and at understanding the physiological significance of QR2.

Crystal Structure of Quinone Reductase 2 in Complex with Resveratrol,

“…However, the protein cannot refold into the active form in the absence of any metal ions in the refolding buffer. Consistent with our studies, Kwiek et al recently reported that the QR2 enzyme purified from human redblood cells contained partially zinc and partially copper ions (45).…”

“…The catalytic mechanism of QR2 and QR1 is likely very similar, a classic ping-pong mechanism (45). FAD is reduced by accepting a hydride from the reduced nicotinamide to form the FADH 2 -enzyme complex, and subsequently, nicotinamide is released from the active site.…”

“…Recently, it was reported that those quinoline derivatives bind to QR2 with an apparent affinity of about 1 µM (45). Even though these compounds exhibit much lower affinity toward QR2 than resveratrol, it is still noteworthy that a broad spectrum of compounds could potentially affect QR2 enzymatic activity.…”

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2

“…Kinetic studies show that PQ and CQ both inhibit NQO2 in the micromolar range, with K I values of 1.0 and 0.6 M, respectively (25), but PQ exhibits competitive inhibition against the reducing co-factor (NRH), whereas CQ competes with the quinone substrate. This pattern of inhibition can be explained by the ping-pong kinetic mechanism that NQO2 uses.…”

An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics

“…QR2 has a role in the detoxification processes of quinones, specifically the two-electron reduction of menadione by the oxidation of N-alkylated or N-ribosylated nicotinamides (34). Knock-out models of QR2 in mice result in an increased susceptibility to polycyclic aromatic hydrocarbon-induced skin carcinogenesis, whereas inhibition of QR2 using quinacrine resulted in death of malaria-infected red blood cells by altering the redox status of the cell (33,35). Recently a role for QR2 in the tumor necrosis factor apoptotic signaling pathway has emerged in which QR2-deficient keratinocytes were shown to have suppressed survival signals and increased tumor necrosis factorinduced apoptosis (36).…”