The kinetic behavior of xanthine oxidase containing chemically modified flavins.
The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.
Hille
R
R
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Massey
V
V
D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D013816:Thermodynamics; D014969:Xanthine Oxidase / Q000378:metabolism*
D005415:Flavins; D014969:Xanthine Oxidase