The binding of oxidized and reduced nicotinamide--adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase.
The binding of NAD(+) and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD(+) and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3mum. The binding of NAD(+) to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60-70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D005947:Glucose; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D013054:Spectrophotometry, Atomic; D014539:Uridine Diphosphate Sugars
D014539:Uridine Diphosphate Sugars; D009243:NAD; D000429:Alcohol Oxidoreductases; D005947:Glucose