THERMODYNAMICS OF BINDING OF THE CO2-COMPETITIVE INHIBITOR IMIDAZOLE AND RELATED-COMPOUNDS TO HUMAN CARBONIC ANHYDRASE-I - AN ISOTHERMAL TITRATION CALORIMETRY APPROACH TO STUDYING WEAK BINDING BY DISPLACEMENT WITH STRONG INHIBITORS

The visible spectrum of Co(II)-substituted human carbonic anhydrase I (HCA I) complexed with the unique CO2-competitive inhibitor imidazole undergoes a marked alkaline intensification, with a midpoint near pH 8 [Bauer, R., Limkilde, P., & Johansen, J. T. (1977) Carlsberg Res. Commun. 42, 325-339]. This change was first attributed to the ionization of an on displaced water ligand of the active-site metal in a five-coordinate complex. Later proposals favored assigning it to the deprotonation of the bound imidazole itself to give a tetrahedrally coordinated imidazolate anion at high pH. We have determined by isothermal titration calorimetry the pH dependence of the enthalpy of binding of imidazole and its analogues to HCA I and Co(II)HCA I. We devised an indirect strategy where by the enthalpy of binding of the strong sulfonamide inhibitor methazolamide was determined in the absence and presence of a constant high concentration of the competing imidazole or its analogues. The standard enthalpy of binding of deprotonated methazolamide to the ''acid'' form of HCA I and Co(II)HCA I was found to be pH independent over the pH range of 6.5-9.5, as expected. It was also identical for both the zinc (-13.5 +/- 1.1 kcal M-1) and the cobalt (-13.7 +/- 0.4 kcal M-1) forms. The standard enthalpy of binding of neutral imidazole (average value -6.1 +/- 0.8 kcal M-1) surprisingly did not show any marked pH dependence, varying by about 1.1 and 2.6 kcal M-1 for the zinc and cobalt enzymes, respectively. The standard enthalpy of binding of the anionic forms of 1,2,3-triazole (-3.8 +/- 0.2 kcal M-1), 1,2,4-triazole (-6.5 +/- 0.4 kcal M-1), and tetrazole (-5.2 +/-0.5 kcal M-1) was found to be pH independent, but in this case no pH-dependent spectral transition had been expected. The absence of appreciable pH changes in the standard enthalpy of binding of imidazole may be due either to the absence of enthalpically significant structural changes in the complex or to compensating heats of binding and ionization that nearly cancel out.