Kinetics and mechanism of mitomycin C bioactivation by xanthine dehydrogenase under aerobic and hypoxic conditions.
These studies examined the kinetic and mechanistic parameters of mitomycin C (MMC) bioreduction by xanthine dehydrogenase (XDH), an enzyme recently shown to be capable of MMC activation. The bioreduction of MMC by XDH leads to the formation of 2,7-diaminomitosene (2,7-DM) under both aerobic and hypoxic conditions, with greater 2,7-DM formation observed under hypoxic conditions. The XDH-induced formation of 2,7-DM is pH dependent with increasing formation as the pH is varied from 7.4 to 6.0. In this study, the kinetics of MMC bioreduction by XDH was assessed under aerobic and hypoxic conditions and at pH 7.4 and 6.0. MMC interaction with XDH was also assessed by monitoring the ability of MMC to inhibit XDH-mediated uric acid and NADH formation. The ability of xanthine to serve as reducing equivalents for MMC reduction was also measured. Aerobically but not hypoxically, MMC reduction by XDH followed Michaelis-Menten kinetics. Kinetic constants calculated under aerobic conditions suggested that the pH-dependent increase (pH 6.0 > pH 7.4) in MMC activation by XDH is due to an approximately 2-fold decrease in the Km and a 2-fold increase in the Vmax at pH 6.0. Stimulation of uric acid formation and decreases in NADH formation by XDH in the presence of MMC suggest that MMC interaction with XDH may occur at the NAD(+)-binding region of the enzyme. The ability of xanthine to serve as reducing equivalents for MMC conversion to 2,7-DM also supports the hypothesis that MMC reduction is occurring at the NAD+ site.
Gustafson
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Department of Nutrition, University of Nevada, Reno 89557-0132.
Pritsos
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CA
D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D001711:Biotransformation; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008819:Mice, Nude; D016685:Mitomycin / Q000493:pharmacokinetics* / Q000494:pharmacology; D009243:NAD / Q000096:biosynthesis / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine; D014968:Xanthine Dehydrogenase / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism
D014970:Xanthines; D009243:NAD; D019820:Xanthine; D014527:Uric Acid; D016685:Mitomycin; D014968:Xanthine Dehydrogenase