Molecular Characterization of Binding of Substrates and Inhibitors to DT-Diaphorase: Combined Approach Involving Site-Directed Mutagenesis, Inhibitor-Binding Analysis, and Computer Modeling

Abstract

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.

Crystal Structure of Quinone Reductase 2 in Complex with Resveratrol,

“…After centrifugation, the protein contents in the supernatants were normalized by BioRad protein assays. The enzymatic activity assays were performed as described (27) with modification, except the total NAD(P)H-dependent quinone reductase assays, which were adopted from Chen et al (28). Briefly, glucose-6-phosphate dehydrogenase activity is measured by the reduction of NADP.…”

NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme The GenBank accession number for the glQR1-encoding sequence reported in this paper is AF321405.

“…increase in catalytic efficiency for NADH, in the presence of naphthoquinone compared with menadione, resulted mainly from a much larger effect on the K app m for NADH (12n5-fold higher than that for NADPH), and to a lesser extent in the k cat value (1n9-fold lower). glQR1 had a much higher affinity for menadione, with NADH as donor, than the human, rat and mouse enzymes (K app m l 2n7p0n3 µM, 2n5p0n1 µM and 4n3p0n4 µM, respectively), while its affinity for NADH was lower (K app m l 220p10 µM, 110p10 µM and 210p20 µM, respectively) (Chen et al, 1999). glQR1 was also able to reduce several alternative electron acceptors with NADPH (Table 3) or NADH (not shown) as electron donor.…”

“…However, H161 of QR1 is G. lamblia NADPH : quinone oxidoreductase substituted in glQR1 by A122, which cannot function in the charge relay suggested. H161 participates in the catalytic reaction and in fact the k cat for a mutant hH161Q is only 8 % of the wild-type hQR1, but it does not interrupt the two-electron transfer reduction of menadione (Chen et al, 1999).…”

Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

“…Studies of NQO1 structure and function have shown that the NAD(P)H and the quinone binding sites of enzymes have a significant overlap (25), and the enzymatic mechanism is a typical ping-pong reaction. In this study, two coumarin derivatives (warfarin and 6,7-dihydroxycoumarin) (26,27), dicoumarol (28), and the reactive dye Cibacron Blue (Reactive Blue-2 (RB-2)) (29,30), which compete with NAD(P)H for binding to NQO1, were tested to see whether these compounds inhibit Ndi1. The structures of these inhibitors are shown in Fig.…”