In contrast to the analogous reaction with xanthine the anaerobic rapid reduction of xanthine oxidase by excess lumazine (2,4-dihydroxypteridine) displays markedly biphasic kinetics at all wavelengths. The fast phase proceeds at a rate (30 s('-1)) approximately a factor of 100 faster than that of the slow phase and about 50 times more rapid than catalysis. During the rapid phase the decrease in absorbance at 450 nm is approximately equal to the corresponding increase at 650 nm ((DELTA)(epsilon) = 6400 M('-1)cm('-1)). The presence of the rapid increase in absorbance at 650 nm during the analogous reduction of deflavo-xanthine oxidase demonstrates that this spectral component is not a function of the flavin. The slow phase shows a decrease in absorbance at all wavelengths proceeding at a rate of 0.3 s('-1).
At the conclusion of this anaerobic reduction with lumazine a net increase in absorbance above 600 nm is observed. The long wavelength absorbance results from the interaction of the end-products of the anaerobic reduction, i.e. Mo(IV) and violapterin (2,4,7-trihydroxypteridine), which gives rise to a charge transfer complex. Circular dichroism studies demonstrate that the charge transfer complex is optimically active. A linear Scatchard plot obtained for the titration of dithionite-reduced enzyme with violapterin yields an apparent dissociation constant of 7.9 (mu)M for the reduced enzyme-product complex. However, the complementary rapid kinetic study of this enzyme-product interaction displays a biphasic time course-indicating that this binding process proceeds by a complex mechanism.
Mixing dithionite-reduced xanthine oxidase with lumazine results in the formation of a charge transfer complex similar to that found with violapterin. No other pteridine tested displays this long-wavelength absorption when mixed with the dithionite-reduced enzyme although a rapid increase in absorbance at 650 nm is observed during the reduction of xanthine oxidase by pterin or xanthopterin. Inactivating the enzyme with either cyanide or allopurinol prevents the formation of the charge transfer absorbance.
The anaerobic reduction of xanthine oxidase by lumazine can be observed by both absorbance and fluorescence spectroscopy. Following the reduction of the enzyme at 450 nm, the formation and subsequent breakdown of the enzyme-product complex at 650 nm, and monitoring the conversion of lumazine to violapterin via fluorescence the kinetics of the reaction can be defined. There appears to be a good correlation between these four optical probes. At pH 8.3 the formation of the enzyme-product complex occurs at a rate of 51 s('-1) while the subsequent breakdown proceeds at 0.4 s('-1). The rate of the former step depends on the absolute concentration of the substrate and an apparent dissociation constant for the enzyme-substrate complex of 34 (mu)M is obtained. The rate of the latter process is independent of the concentration of the substrate although this rate does appear to be affected by the stoichiometry of the reaction. These results are consistent with the model of Olson, J.S., Ballou, D., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382 formulated for the analogous study with xanthine. Finally, there exists a reasonably good correlation between the kinetic parameters obtained from the rapid kinetic studies and those determined for the complementary steady-state results.
The effect of pH on the reaction of xanthine oxidase with lumazine mimics that found for the analogous reaction with xanthine. These studies lead to a more general description of the reaction mechanism of xanthine oxidase.