Linkage of subunit interactions, structural changes, and energetics of coenzyme binding in tryptophan synthase.
The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods. At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps. Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs. time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain. The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation. The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions. Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs. On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS)
D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000378:metabolism*
D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase