1-substituted phthalazines as probes of the substrate-binding site of mammalian molybdenum hydroxylases.
The interaction of a series of 1-substituted phthalazine derivatives with partially purified aldehyde oxidase from rabbit, guinea-pig and baboon liver, and with bovine milk xanthine oxidase, has been investigated. Of the 18 compounds examined, rabbit liver aldehyde oxidase metabolized 10, whereas guinea-pig and baboon liver enzyme oxidized 13 and 14, respectively. Where metabolites were characterized, oxidation was shown to occur at position four of the phthalazine ring. Km values ranged from 0.003 to 1.8 mM. In contrast, most compounds were competitive inhibitors of bovine milk xanthine oxidase with Ki values ranging from 0.015 to 1.3 mM; the cationic derivative 2-methylphthalazinium iodide was oxidized to 2-methyl-1-phthalazinone by both aldehyde oxidase and, with a much reduced affinity, by xanthine oxidase. In terms of structure-metabolism relationships, Vmax values were relatively insensitive to the electronic effects of substituents, but a trend for the more lipophilic derivatives to show increased affinities (Km and Vmax/Km) towards aldehyde oxidase could be seen. However, calculations of molecular size revealed a species-dependent cut-off threshold above which compounds were not metabolized. Results suggest that the relative size of the active site for hepatic aldehyde oxidase is in the order baboon greater than guinea-pig greater than rabbit, and that in spatial terms the active site of bovine milk xanthine oxidase is similar to that of baboon liver aldehyde oxidase. Thus, the binding site of rabbit liver aldehyde oxidase, a widely used source of the oxidase, is apparently more restricted than in some other species.
Beedham
C
C
School of Pharmacy, University of Bradford, U.K.
Bruce
S E
SE
Critchley
D J
DJ
Rance
D J
DJ
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D015335:Molecular Probes; D008982:Molybdenum; D010084:Oxidation-Reduction; D010215:Papio; D010793:Phthalazines / Q000378:metabolism* / Q000494:pharmacology; D011724:Pyridazines / Q000378:metabolism*; D011817:Rabbits; D013055:Spectrophotometry, Infrared; D013379:Substrate Specificity; D014969:Xanthine Oxidase / Q000378:metabolism
D015335:Molecular Probes; D010793:Phthalazines; D011724:Pyridazines; D008982:Molybdenum; D014969:Xanthine Oxidase; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase