Kinetics, mechanism and inhibition of human quinone reductase type-2 (HQR2)

Recombinant hQR2 is a flavoenzyme that catalyzes the same reaction of FAD-mediated hydride transfer as quinone reductase type-1 (QR1 or DT-Diaphorase, EC 1.6.99.2), except that QR2 utilizes only nonphosphorylated N-nbosyl and W-alkyl dihydronicotinamides as hydride donors, but not NADH, NADPH or NMNH (Zhao et al., PNAS 94, 1669-1674, 1997). hQR2 has been efficiently over-expressed in E. coli, purified to homogeneity to a specific activity of 248 umolmin-mg and recently crystallized. hQR2 (231 aa) can be viewed as a truncated analog of hQRl (274 aa) in which the C-terminal 43 aa are absent and there is 49% identity. This tail is important in binding the pyrophosphate portion of N AD(P)H according to modeling based on the x-ray crystal structure of rat QR1 (Li et al, PNAS 92, 8846-8850, 1995). Truncation of the lysine-rich 43 aa tail results in high affinity for hydrophobic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene, whereas dicumarol and Cibacron blue, highly potent inhibitors of QR1, only weakly inhibit hQRZ High affinity binding (KD = 50 nM) of benzo(a)pyrene to hQR2 was demonstrated by fluorescence quenching. Inhibition constants for benzo(a)pyrene are Kjc = 70 nM with respect to V-methyldihyronicotinamide and Kiu = 90 nM with respect to menadione.