TY - JOUR
T1 - Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse
AU - Chen, Shiuan
AU - Knox, Richard
AU - Wu, Kebin
AU - Deng, Paulis S.K.
AU - Zhou, Dujin
AU - Bianchet, Mario A.
AU - Amzel, L. Mario
PY - 1997/1/1
Y1 - 1997/1/1
N2 - DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone- acceptor) 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 significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs 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 rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amine acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural 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 region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.
AB - DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone- acceptor) 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 significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs 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 rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amine acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural 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 region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.
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U2 - 10.1074/jbc.272.3.1437
DO - 10.1074/jbc.272.3.1437
M3 - Article
C2 - 8999809
AN - SCOPUS:0031026290
SN - 0021-9258
VL - 272
SP - 1437
EP - 1439
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 3
ER -