Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

Abstract

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear. On the basis of these results an empirical rate equation was deduced from a series of slope and intercept plots. Kinetic constants were evaluated by the method of weighted least squares using an IBM 360 digital computer. The standard error of the calculated reciprocal velocity was only 4.7% of the weighted average of the observed reciprocal velocity. No term in this empirical equation could be omitted or rearranged and attain the same degree of agreement. This nonlinear rate equation can be interpreted using random substrate addition and product-release kinetic models with independent catalytic sites and without postulating second sites.