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 K_m, K_i 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 (K_i= 9 μM), 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 μM and a partial competitive inhibitor above this concentration, which implies the presence of a second binding site.