Microcalorimetry and high performance liquid chromatography were used to conduct a thermodynamic investigation of the biochemical reaction chorismate(aq) = isochorismate(aq). This reaction occurs at the branch point of the chorismate metabolic pathway that leads to the synthesis of enterobactin. Isochorismate synthase, the enzyme that catalyzes this reaction, was prepared for this study by using molecular biology techniques. The equilibrium and calorimetric measurements were performed at T = 298.15 K and at the respective pHs 7.34 and 6.93. For the chemical reference reaction chorismate(2-)(aq) = isochorismate(2-)(aq), the equilibrium constant K = (0.84 +/- 0.04) and the standard molar enthalpy of reaction Delta(r)H(m)(O) = -(0.81 +/- 0.27) kJ . mol(-1) at T = 298.15 K and ionic strength I-m = 0. Under approximately physiological conditions, die apparent equilibrium constant K' = 0.83 and the standard transformed gibbs energy change Delta(r)G'(O)(m) = 0.48 kJ . mol(-1) for the overall biochemical reaction.