Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus.
Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.
D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013440:Sulfides / Q000032:analysis; D014968:Xanthine Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D013440:Sulfides; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases