Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study.
Rat liver deflavoxanthine dehydrogenase has been prepared by incubating native enzyme with calcium chloride. On reconstitution with FAD, about 85% of the original activity is recovered, all which is the O2-dependent type. In contrast, when dithiothreitol-treated deflavoenzyme is incubated with FAD, the recovery of activity is almost the same as above, but most of the recovered activity is of the NAD-dependent type. Deflavoenzyme with or without previous treatment with dithiothreitol was also reconstituted with two artificial FAD analogues, 8-mercapto-FAD and 6-OH-FAD. The difference spectra between the reconstituted enzymes and the initial deflavoenzyme indicate that, in each case, the FAD analogue is bound in its neutral form in dithiothreitol-treated enzyme, whereas it is bound in the anionic form in enzyme without previous dithiothreitol treatment. Furthermore, the protonated forms can be converted into the anionic forms on storage with a concomitant change of activity from the NAD-dependent to the O2-dependent type. This clearly indicates different environments around FAD in the two types of enzyme protein, which are shown to be interconvertible through oxidation-reduction of enzyme cysteinyl residues.
Saito
T
T
Department of Biochemistry, Yokohama City University School of Medicine, Japan.
Nishino
T
T
Massey
V
V
D000818:Animals; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology; D051381:Rats; D013053:Spectrophotometry; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
D007527:Isoenzymes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases; D010100:Oxygen; D004229:Dithiothreitol