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

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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 β-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 kcat values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 ± 1, 23 ± 0, 5 ± 0 and 8 ± 2 × 103 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.