title: Determination of the energetics of the UDP-glucose pyrophosphorylase reaction by positional isotope exchange inhibition.
abstract: A method has been developed for obtaining qualitative information about enzyme-catalyzed reactions by measuring the inhibitory effects of added substrates on positional isotope exchange rates. It has been demonstrated for ordered kinetic mechanisms that an increase in the concentration of the second substrate to add to the enzyme will result in a linear increase in the ratio of the chemical and positional isotope exchange rates. The slopes and intercepts from these plots can be used to determine the partitioning ratios of binary and ternary enzyme complexes. The method has been applied to the reaction catalyzed by UDP-glucose pyrophosphorylase. A positional isotope exchange reaction was measured within oxygen- 18-labeled UTP as a function of variable glucose 1-phosphate concentration in the forward reaction. In the reverse reaction, a positional isotope exchange reaction was measured within oxygen- 18-labeled UDP-glucose as a function of increasing pyrophosphate concentration. The results have been interpreted to indicate that the interconversion of the ternary central complexes is fast relative to product dissociation in either direction. In the forward direction, the release of UDP-glucose is slower than the release of pyrophosphate. The release of glucose 1-phosphate is slower than the release of UTP in the reverse reaction.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station 77843.
authors.forename: L S
authors.initials: LS
authors.lastname: Hester
authors.forename: F M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010103:Oxygen Isotopes; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D010103:Oxygen Isotopes; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014544:Uridine Triphosphate