- Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: Implication to drug pharmacokinetics
-
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.
- Tai,Hwang
-
experimental part
p. 69 - 78
(2012/01/05)
-
- Enzymatic inhibition assay for fluorometric determination of allopurinol and oxipurinol in serum and urine
-
An enzymatic inhibition assay for the xanthine oxidase (XOD) inhibitors allopurinol and oxipurinol is described. 2-Amino-4-hydroxypteridine is used as sensitive fluorogenic substrate, which is oxidized to highly fluorescent isoxanthopterin by XOD. Increasing concentrations of allopurinol (Ap) or oxipurinol (Ox) prevent conversion of 2-amino-4-hydroxypterdine to isoxanthopterin. Assay conditions of XOD-inhibition are different for Ap and Ox. In the presence of xanthine both Ap and Ox inhibit XOD to the same degree; absence of xanthine results in inactivation by Ap only. Thus rapid and convenient determination of Ap and Ox is possible without prior separation of the substances. The limit of detection is about 0.5 nmol/ml (50 μg/ml) in serum and 25 nmol/ml (2.5 μg/ml) in urine.
- Neubert,Besenfelder,Koch
-
p. 1857 - 1859
(2007/10/02)
-