Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase.
The binding of NADH to uridine diphosphate glucose dehydrogenase has been examined by equilibrium dialysis. There is an absolute requirement for the presence of UDP-glucose for the binding of NADH. Other analogs such as UDPxylose, UDPgalactose and UDPglucuronic acid cannot replace UDPglucose as an effector of NADH binding. UDPxylose competes with UDPglucose for the UDP-sugar-binding site, and in so doing releases the bound NADH. The binding of NADH to UDPglucose dehydrogenase in the presence of UDPglucose reaches a saturation limit of 3 mol NADH bound per enzyme hexamer, and displays positive cooperativity, Hill number = 1.34. The effects of UDP-sugars on the fluorescence of UDPglucose dehydrogenase derivatized at the catalytic sites with a fluorophore have also been studied. Two classes of UDPxylose-binding site have been detected. One class has high affinity (Kdiss = 3 microM, determined by equilibrium dialysis) but does not affect fluorophore fluorescence, and the other has lower affinity (Kdiss = 120 microM) and leads to red-shifted fluorescence quenching, presumably by effecting exposure of the fluorophore to solvent. The high-affinity sites are identified as the UDP-sugar subsites of the underivatized catalytic sites, and the low-affinity sites as UDP-sugar subsites of the fluorophore-labeled catalytic sites.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D002417:Cattle; D005456:Fluorescent Dyes; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009282:Naphthalenesulfonates; D013050:Spectrometry, Fluorescence; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism* / Q000494:pharmacology; D014540:Uridine Diphosphate Xylose / Q000378:metabolism
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014539:Uridine Diphosphate Sugars; D009243:NAD; D014540:Uridine Diphosphate Xylose; C006421:1,5-I-AEDANS; D002237:Carbohydrate Dehydrogenases; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014532:Uridine Diphosphate Glucose