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

These studies examined the kinetic and mechanistic parameters of mi tomycin C (MMC) bioreduction by xanthine dehydrogenase (XDH), an enzyme recently shown to be capable of MMC activation. The bioreduc tion 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 forma tion 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 reduc tion 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 A'm and a 2-fold increase in the \ ,„.,,, at pH 6.O. 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.