Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase.
Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D014443:Tyrosine; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
D009243:NAD; D014443:Tyrosine; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases