Abstract
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.
Highlights
Terin, the structure of which has been proposed and recently reviewed [8,9]
The sulfur atom necessary for catalytic activity has been shown by x-ray absorption spectroscopy to he present as a Mo=S group [14,15,16,17,18]
As the enzyme is involved in the final steps of uric acid production, an inhibitor of xanthine oxidase is useful for remedy of hyperuricemia or gout [19]
Summary
Terin, the structure of which has been proposed and recently reviewed [8,9]. Two electrons are transferred from xanthine to Mo(VI), reducing the metal to Mo(IV) while another substrate, NAD or oxygen, reacts with FAD; the catalytic cycle necessarily entails intramolecular electron transfer. Steady State Analysis ofInhibition ofXanthine Oxidase with BOF-4272-The inhibitory effect of allopurinol on XO is known to be time dependent; the activity decreases gradually after mixing the enzyme with allopurinol.
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