Differences in redox and kinetic properties between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase.
Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.
Saito
T
T
Department of Biochemistry, Yokohama City University School of Medicine, Japan.
Nishino
T
T
D000818:Animals; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D014968:Xanthine Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D009243:NAD; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases; D010100:Oxygen