Specificity of xanthine oxidase for nitrogen heteroaromatic cation substrates.
A variety of pyridinium, quinolinium, and benzoquinolinium cations have been investigated as potential substrates for milk xanthine oxidase at pH 9.9 and (or) pH 10.6. Steady-state kinetic parameters (kc, Km and (or) kc/Km) have been evaluated for all substrates which are enzymically oxidized. Simple N-alkyl pyridinium cations are neither substrates nor inhibitors, although N-aryl pyridinium cations are slowly oxidized to the 4-pyridinones. N-Methylpyridinium cations bearing 3-CONH2, 3-CONHCH3, 3-COCH3, 3-CO2- or 3-CN substituents are readily oxidized at C-6 and this suggests an important hydrogen-bonding interaction between an enzyme donor and the C-3 carbonyl substituent. A variety of N-methylquinolinium cations bearing C-6 substituents are enzymically oxidized at C-2. Analogous substituent effects on kc/Km for these 6-substituted 1-methylquinolinium cations and the corresponding 1-(substituted phenyl)-pyridinium cations is suggestive of the relative productive binding orientations of these two classes of substrate in the active site. N-Methylbenzoquinolinium and 1,10-phenanthrolinium cations are the best cationic substrates found to date, and suggest a relatively large active-site region for the reducing substrate, and important hydrophobic interactions between enzyme and substrate. The overall enzymic specificity observed for these cationic substrates allows a mapping of the general features of the reducing substrate binding site of this enzyme.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D008956:Models, Chemical; D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; D013379:Substrate Specificity; D014969:Xanthine Oxidase / Q000378:metabolism*
D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; D014969:Xanthine Oxidase