KINETICS AND MECHANISM OF MITOMYCIN-C BIOACTIVATION BY XANTHINE DEHYDROGENASE UNDER AEROBIC AND HYPOXIC CONDITIONS

These studies examined the kinetic and mechanistic parameters of mitom ycin C (MMC) bioreduction by xanthine dehydrogenase (XDH), an enzyme r ecently 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 b oth aerobic and hypoxic conditions, with greater 2,7-DM formation obse rved 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 asses sed under aerobic and hypoxic conditions and at pH 7.4 and 6.0. MMC in teraction with XDH was also assessed by monitoring the ability of MMC to inhibit XDH-mediated uric acid and NADH formation. The ability of x anthine to serve as reducing equivalents for MMC reduction was also me asured. 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 i n 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 p resence 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 h ypothesis that MMC reduction is occurring at the NAD+ site.