title: Methylated 7-deazahypoxanthines as regiochemical probes of xanthine oxidase.
abstract: 7-Deazahypoxanthine was found to be oxidised by cow's milk xanthine oxidase exclusively at carbon 2. The resulting 7-deazaxanthine is a strong inhibitor of the enzymatic reaction. This offers a possibility for determining the structural requirements of ligand binding separately for the first step. All the monomethyl isomers of 7-deazahypoxanthine were tested as probes by measuring their Km, Ki and V values. While the N-3-methyl and C-7-methyl isomers are still processed, the N-9-methyl and 6-O-methyl isomers are bound as inhibitors to the active site. The N-1-methyl compound is neither an inhibitor nor a substrate. This demonstrates that HN(1) and O = C(6) are essential for the binding. Replacement of O = C(6) by S = C(6) changes the substrate into a strong inhibitor (Ki = 9 microM), implying that the electron transfer to the enzyme is hindered. Methylation of the thioxo group (S =) reduces the inhibition significantly. In contrast to 7-deazahypoxanthine, 2-thioxo-7-deazaxanthine is an activator at concentrations below 87 microM and a partial competitive inhibitor above this concentration, which implies the presence of a second binding site.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007042:Hypoxanthines / Q000378:metabolism* / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008745:Methylation; D008892:Milk / Q000201:enzymology; D010084:Oxidation-Reduction / Q000187:drug effects; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D007042:Hypoxanthines; D014969:Xanthine Oxidase