Human and bovine xanthine oxidases. Inhibition studies with oxipurinol.
Oxipurinol inhibited human xanthine oxidase and bovine xanthine oxidases by very similar mechanisms. It bound to an electronically reduced form of human xanthine oxidase in a manner similar to that previously discerned from its interactions with the bovine enzyme [review article: Spector, Biochem. Pharmac. 26, 355 (1977)]. Xanthine was a good source for the reducing equivalents because it did not compete with oxipurinol for binding to reduced enzyme. The inhibition of the rate of urate production progressively increased with time. Studies of the effect of the concentration of oxipurinol on the rate constant of the development of this inhibition revealed that a complex was rapidly formed between oxipurinol and reduced bovine or human xanthine oxidases (KD of about 8 microM). At 37 degrees these complexes were converted to stable complexes at a maximum rate of about 1.6 min-1. The rate constant was highly temperature dependent with an energy of activation of 30 kcal/mole (cf. 13 kcal/mole for the energy of activation for catalysis). These data support the earlier conclusions that the formation of stable complexes probably reflects a massive rearrangement of the initial complexes. The isolated oxipurinol-xanthine oxidase complexes spontaneously reverted to active enzyme with a rate constant of 0.02 min-1 at 37 degrees. The energy of activation for the "reactivation" was similar to that for the formation of the stable complexes. The rates of "reactivation" could be stimulated by high concentrations of xanthine: 2.4-fold at 50 microM and 3.4-fold at 100 microM. The constant for the overall inhibition by oxipurinol was approximately 100 nM with both enzymes.
D000818:Animals; D002417:Cattle; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D010117:Oxypurinol / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors*
D011743:Pyrimidines; D014969:Xanthine Oxidase; D010117:Oxypurinol