THE EFFECT OF TEMPERATURE ON THE BINDING OF SULFONAMIDES TO CARBONIC-ANHYDRASE ISOENZYME-I, ISOENZYME-II, AND ISOENZYME-IV

We report the effect of temperature on the equilibrium dissociation constants (K-I) for a series of six sulfonamides binding to three carbonic anhydrase (CA) isoenzymes (I, II, and IV). K-I values obtained at 0 degrees, 15 degrees, and 23 degrees under conditions of nearly constant and low substrate (CO2) concentration were used to calculate enthalpy and entropy changes associated with sulfonamide binding as well as to provide estimates of inhibitory potency of sulfonamides at 37 degrees. We studies four classic sulfonamides (methazolamide, benzolamide, ethoxzolamide, and sulfanilamide) and the novel sulfonamides MK-507 (dorzolamide) and CF3SO2NH2. In all cases, the K-I was observed to increase with increasing temperature, which is consistent with a negative enthalpy of sulfonamide binding. The extrapolated increase in K-I over the 0-37 degrees temperature range varied from 4-fold for sulfanilamide binding to CAI to 14-fold for CF3SO2NH2 binding to CA IV, corresponding to binding enthalpy values of -7.2 to -11.7 kcal/mol. For CA II and I, entropy changes associated with sulfonamide binding were in general modest and ranged from -5.3 to +4.1 entropy units (eu) for five of the compounds tested. In contrast, ethoxzolamide binding was associated with a relatively large positive entropy change. Also, the variatione in k(on) and k(off) with temperature were studied for three sulfonamides binding to CA II. The association rate constants for methazolamide, benzolamide, and ethoxzolamide binding showed increases of e-fold or less, whereas dissociation constants increased 3-9-fold over the range of 0-37 degrees. Thus, the temperature effect in increasing K, is in large part due to a faster rate of sulfonamide dissociation. Apparent activation parameters at 23 degrees for k(on) were Delta H double dagger = -2.35 to 3.8 kcal/mol, Delta G double dagger = 7.3 to 8.6 kcal/mol, and Delta S double dagger = -16.2 to -32.7 entropy unit. For k(off), the corresponding values were Delta H double dagger = 5.6 to 14.5 kcal/mol, Delta G double dagger = 19.0 kcal/mol, and Delta S = -14.8 to -45.7 entropy units.