Regulation of Xanthine Oxidase Activity by Substrates at Active Sites via Cooperative Interactions between Catalytic Subunits: Implication to Drug Pharmacokinetics

L.      A. Tai; K.      C. Hwang

Abstract

Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a “substrate inhibition” pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a “substrate activation” pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s-1 to 10.5 s-1, and converts the V-[S] plot from “substrate inhibition” pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.

Keywords: Cooperativity, 6-formylpterin, regulation, substrate inhibition, substrate activation, xanthine oxidase, enzyme kinetics, kinetic behavior, enzymes, concentration, velocity, Michaelis-Menten, hyperbolically, methylamine dehydrogenase, homo-tetrameric, binding affinity, xanthine, oxygen, superoxide, nitric oxide, catalytic subunits, buffer solution, pH, hyperbolic saturation, relative magnitude, transition point, hyperbolic saturation region, reactivity, hetero-substrates, homo-substrates, uric acid, dissociation constant, isoxanthopterin, half life, nitric oxide formation, drug design, catalytic activity, dissociation rate constants