Thermodynamic contributions of various metal-nucleotide complexes binding to yeast phosphoglycerate kinase

by 1977- Holzberger, Randall Allen

The purpose of this study was to determine the thermodynamic parameters of various enzyme-substrate complexes of Yeast Phosphoglycerate Kinase by using Isothermal Titration Calorimetry (ITC). The addition of metal-nucleotide substrate to a PGK solution elicited an exothermic response ranging from 0.5 kilocalories to 2.8 kilocalories. Titrations of MgATP and MgADP into PGK yielding binary complexes generated a greater amount of heat than titrations of MgADP into PGA-PGK and MgAMP-PCP into PGA-PGK yielding ternary complexes. The entropic contribution of the ternary complex formations was significantly greater than binary complex formations. Although entropic and enthalpic contributions varied, the Gibbs free energy remained relatively constant for the binary and ternary enzyme-substrate complexes. This data suggests that the entropic contribution becomes more dominant when PGA was added to form ternary complex of enzyme and ligand. The titration of sugar substrate, 3-phosphoglyceric acid (PGA), into a PGK solution yielded an endothermic signal. The magnitude of the thermodynamic parameters remained elusive however. Numerous different regulatory molecules, which share many of the ionic characteristics of PGA, have been demonstrated to bind PGK in as many as six different regions within the catalytic core and along the periphery of the protein. As a result, titrations of PGA into PGK did not yield interpretable thermodynamic data. Enthalpy is a global entity encompassing the heat change of the entire system, and iii consequently each titration likely included PGA binding at the desired active site in addition to a combination of other binding events at different areas of the enzyme. Finally, the addition of sulfate to the various PGK complexes increased the dissociation constant of most of the substrates from the binary and ternary enzyme-substrate complexes and altered all thermodynamic properties of these complexes. iv