Mechanism of inhibition of xanthine oxidase with a new tight binding inhibitor.
The mechanism of inhibition of milk xanthine oxidase and xanthine dehydrogenase by the tight binding inhibitor, sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5- triazine-4-olate monohydrate (BOF-4272), was studied after separation of the two isomers. The steady state kinetics showed that the inhibition by these compounds was a mixed type. One of the isomers had a Ki value of 1.2 x 10(-9) M and a Ki' value of 9 x 10(-9) M, while the other isomer had a Ki value of 3 x 10(-7) M and a Ki' value of 9 x 10(-6) M. Spectral changes were not observed by mixing either the oxidized or reduced form of the enzyme with BOF-4272. The stopped-flow study and the effects of BOF-4272 on various substrates showed that BOF-4272 bound to the xanthine binding site of the enzyme. Kd values of the enzyme and one of the isomers, which has a higher affinity for the enzyme, were also found to be 2 x 10(-9) M for the active form of the enzyme and 7 x 10(-9) M for the desulfo-form using fluorometric titration, and the binding has stoichiometry of 1:1. The inhibitor could not bind to the enzyme when the enzyme was previously treated with oxipurinol.
Okamoto
K
K
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Nishino
T
T
D007536:Isomerism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D014227:Triazines / Q000378:metabolism / Q000494:pharmacology*; D019820:Xanthine; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D014227:Triazines; D014970:Xanthines; D009243:NAD; D019820:Xanthine; C072977:BOF 4272; D014969:Xanthine Oxidase