MOLECULAR-BASIS OF THE CATALYTIC DIFFERENCES AMONG DT-DIAPHORASE OF HUMAN, RAT, AND MOUSE

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acce ptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is signifi cantly more effective in activating these drugs than the human and mou se enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal pr odrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the ra t sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsib le for the catalytic differences between the rat and the human (and mo use) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wildtype human enzyme, and the human mutant Q10 4Y behaved like the wild-type rat enzyme in their ability to reductive ly activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrob enzamide). The study also confirms the conclusion of the x-ray structu ral analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding re gion of the nicotinamide portion of NAD(P)H. This structural informati on is very important for designing suitable drugs and approaches for h uman cancer chemotherapy mediated by DT diaphorase.