The Journal of Biological Physics and Chemistry

Differential substrate affinity, kinetics and electron transfer reaction displayed by xanthine and 6-thioxanthine as substrates of milk xanthine oxidase

Hemlata Tamta, Sukirti Kalra, Giddabasappa Channabasava Swamy Anand and Anup Kumar Mukhopadhyay

Xanthine oxidase (E.C.1.1.3.22) is a metallo redox enzyme, which catalyses the oxidative hydroxylation of hypoxanthine or xanthine to uric acid along with the formation of reactive oxygen species like superoxide (1^- product) and hydrogen peroxide (2e^- product). A thiopurine-based antileukaemic drug, 6-mercaptopurine, is also a substrate of xanthine oxidase. 6-thioxanthine is an intermediate of the hydroxylation reaction of 6-mercaptopurine catalysed by xanthine oxidase. In this work, we have compared the kinetics and the one or two electron distribution alternatives between the natural substrate xanthine and the thiopurine substrate 6-thioxanÂthine. From the results of three different experimental approaches, namely comparing K_m values, a two-substrate competition experiment and comparing allopurinol IC₅₀ values, it has been conclusively shown that the affinity of 6-thioxanthine for xanthine oxidase is stronger than that of xanthine. Experiments on the turnover of product formation showed that even though xanthine oxidase has less affinity for the natural substrate xanthine than for 6-thioxanthine, the turnover of uric acid formation from xanthine is faster compared to that of 6-thiouric acid formation from 6-thioxanthine. Temperature was found to profoundly modulate the K_m and V_max values for both substrates. The different affinity and turnover kinetics of these two substrates for xanthine oxidase was also reflected in hydrogen peroxide and superoxide formation. It was found that the initial rates of either hydrogen peroxide or superoxide formation are slower in the case of 6-thioxanthine as substrate than in the case of xanthine. However, the total amount of superoxide generated enzymatically from 6-thioxanthine was ultimately higher than that from xanthine at the four different temperatures studied. On the contrary, the total quantity of hydrogen peroxide formed enzymatically from xanthine was higher than that from 6-thioxanthine. Stronger binding of 6-thioxanthine with the enzyme is associated with the formation of higher amounts of superoxide, but inversely, stronger substrate binding to xanthine oxidase is associated with a slower catalytic turnover of 6-thioxanthine into 6-thiouric acid relative to xanthine.

Keywords: differential kinetics, differential substrate affinity, electron transfer, enzyme energetics, IC₅₀, temperature effect, two-substrate competition, xanthine oxidase