Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase.
A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D008024:Ligands; D008099:Liver / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D014500:Uracil Nucleotides / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D008024:Ligands; D014500:Uracil Nucleotides; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose