Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit‐binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (ΔH°) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (ΔG° = −12.6 kcal/mol for E3 and = −12.9 kcal/mol for E1 at pH 7.4 and 25°C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (ΔH° = +2.2 kcal/mol) and a large, positive entropy change (TΔS° = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (ΔH° = −8.4 kcal/mol) and a less positive entropy change (TΔS° = +4.5 kcal/mol). Values of ΔCp of −316 cal/molK and −470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the ΔCp calculated from the nonpolar surface area buried in the crystal structure of the E3‐PSBD complex. In this instance, a large negative ΔCp is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (Tm) of E3 increased from 91°C to 97.1°C when it was bound to PSBD, and that of E1 increased from 65.2°C to 70.0°C. These high Tm values eliminate unfolding as a major source of the anomalous ΔCp effects at the temperatures (10–37°C) used for the ITC experiments.