title: Thermodynamic analysis of the activation of glycogen phosphorylase b over a range of temperatures.
abstract: Equilibrium dialysis and isothermal microcalorimetry experiments have been carried out to characterize the thermodynamics of the binding of AMP to glycogen phosphorylase b (EC 2.4.1.1) at pH 6.9 over the temperature range of 25-35 degrees C. Thermal titrations were performed at each temperature in various buffer systems, which have afforded the calculation of the number of protons exchanged when the AMP binds to each site in the protein. Thermodynamic parameters were obtained for the binding of AMP to the two nucleotide and the two inhibitor sites of the dimeric enzyme. The former show positive cooperativity while the latter behave as independent binding sites. A positive delta Cp value was obtained for the AMP binding to the two N sites (1.3 and 1.4 kJ K-1 mol-1), while the delta Cp was negative for the binding to the I sites (-1.9 kJ K-1 mol-1). The application of Sturtevant's method to our data (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240) and their comparison with a similar analysis undertaken with phosphorylase a (Mateo, P. L., González, J. F., Barón, C., Lopez-Mayorga, O., and Cortijo, M. (1986) J. Biol. Chem. 261, 17067-17072) has opened the way to some understanding of the thermodynamics of the allosteric transition in the protein.
authors.affiliation: Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Barón
authors.forename: J F
authors.initials: JF
authors.lastname: González
authors.forename: P L
authors.initials: PL
authors.lastname: Mateo
authors.forename: M
authors.initials: M
authors.lastname: Cortijo
mesh_terms: D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D003956:Dialysis; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010763:Phosphorylase b / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: D010763:Phosphorylase b; D006005:Phosphorylases