Influence of 8-substitutes on the oxidation of hypoxanthine and 6-thioxopurine by bovine milk xanthine oxidase.

1. The influence of 8-substituents was studied on the rate of oxidation of hypoxanthine and 6-thioxopurine by bovine milk xanthine oxidase (EC 1.2.3.2). 2. An 8-methyl group does not alter the rate of oxidation of hypoxanthine materially, but an 8-phenyl substituent reduces it markedly. This is ascribed to inhibition of the tautomerisation process, responsible for substrate activation, prior to oxidation. 3. In contrast, the 8-phenyl group in 3-methyl-8-phenylhypoxanthine enhances the rate, presumably by binding to a hydrophobic site near the enzymaic center. 4. An 8-phenyl group in 6-thioxopurine markedly increases the rate of enzymaic oxidation. Probably the aromatic substituent diverts anion formation to the imidazole ring. In contrast, ionisation of 8-methyl-6-thioxopurine involves the pyrimidine moiety, thus rendering enzymic attack at position 2 more difficult.