Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: Combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling

The molecular basis of the interaction of DT-diaphorase with a cytotoxic ni trobenzamide CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and five inhib itors was investigated with wild-type DT-diaphorase (human and rat) and fiv e mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothes is that Tyr155 and His161 participate in the obligatory two-electron transf er reaction of the enzyme. The catalytic properties of hY155F and hH161Q we re compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutati on disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide pros thetic 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 +/- 6 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catal ytic activity, the hydroxyl group on Tyr155 may not be as important as prop osed. Interestingly, hY155F was found to be 3.3 times more active than the human wildtype DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, w ith the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysi n, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the e nzyme with respect to nicotinamide coenzymes. The binding orientations of d icoumarol, flavones, and phenindone in the active site of DT-diaphorase wer e predicted by results from our inhibitor-binding studies and computer mode ling based on published X-ray structures. Our studies generated results tha t explain why dicoumarol is a potent inhibitor and binds differently from f lavones and phenindone in the active site of DT-diaphorase.