Inhibition Studies of Bovine Xanthine Oxidase by Luteolin, Silibinin, Quercetin, and Curcumin

Abstract

Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally-occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steadystate kinetic studies. We find that luteolin, silibinin, and quercetin are mixed-type inhibitors of the enzyme in vitro and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other non-mechanistic studies, we find that curcumin did not inhibit the activity of purified XO, nor its superoxide production in vitro.Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-containing enzyme that has been studied extensively from a biochemical perspective for more than 80 years. In human physiology, XOR catalyzes the final two steps of purine catabolism, transforming hypoxanthine to xanthine and then xanthine to uric acid by sequential oxidative hydroxylations at C-2 and C-8 of the purine ring, respectively ( Figure 1). 1 Xanthine oxidoreductase exists in two forms. The protein normally exists as a dehydrogenase (xanthine dehydrogenase, XDH), and utilizes NAD + as its final electron acceptor in catalysis. Under certain conditions, most notably ischemia and/or hypoxia, XDH can be converted to an oxidase form (XO), which can no longer reduce NAD + and instead utilizes O 2 exclusively as the terminal electron acceptor in the course of turnover. This conversion may occur either by oxidation of sulfhydryl groups and/or by limited proteolysis. 2, 3 Once in the oxidase form, the enzyme generates significant amounts of H 2 O 2 and superoxide radicals, although XDH can also react with O 2 and generate these reactive oxygen species (ROS). 3 Under normal conditions, however, NAD + effectively competes with O 2 and limits the generation of ROS by the dehydrogenase. 6,7 The specific conformational change responsible for the D to O conversion involves modification of the access channel to the flavin site (with its FAD cofactor) of XOR, eliminating NAD + binding. 3, 4 Once generated, these ROS can interfere with a multitude of cellular functions and processes. Two of the most extensively studied are the integrity of cellular membrane lipids and the interactions of superoxide with the vasodilator NO in circulation. In the latter case, superoxide from xanthine oxidase has been shown to degrade S-nitrothiols, which are considered to be a storage form of NO, as well as converting NO to the vaso-inactive compound peroxynitrite. 8,9 Superoxide has also been shown to reduce H 2 O 2 to form destructive hydroxyl radicals, and even carbonate radicals. 10 * To whom correspondence may be addressed T: (951) XOR is thus potentially a main player in many pathological conditions, and additio...

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