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Kinetics and Mechanism of Mitomycin C Bioactivation by Xanthine Dehydrogenase under Aerobic and Hypoxic Conditions¹

Department of Nutrition and the Cellular and Molecular Pharmacology and Physiology Program, University of Nevada, Reno, Nevada 89557-0132

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 K_m and a 2-fold increase in the V_max 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.

¹ This work was supported by USPHS Grant CA-43660 from the National Cancer Institute and by the Nevada Agricultural Experiment Station.

² Present address: School of Pharmacy, University of Colorado Health Science Center, Denver, CO 80262.

³ To whom requests for reprints should be addressed, at Department of Nutrition, Cell and Molecular Pharmacology Physiology Program, University of Nevada, Mail Stop 142, Reno, NV 89557-0132.

Received 5/ 7/93. Accepted 9/14/93.

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