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 co nstants (K-I) for a series of six sulfonamides binding to three carbon ic anhydrase (CA) isoenzymes (I, II, and IV). K-I values obtained at 0 degrees, 15 degrees, and 23 degrees under conditions of nearly consta nt and low substrate (CO2) concentration were used to calculate enthal py and entropy changes associated with sulfonamide binding as well as to provide estimates of inhibitory potency of sulfonamides at 37 degre es. We studies four classic sulfonamides (methazolamide, benzolamide, ethoxzolamide, and sulfanilamide) and the novel sulfonamides MK-507 (d orzolamide) and CF3SO2NH2. In all cases, the K-I was observed to incre ase with increasing temperature, which is consistent with a negative e nthalpy of sulfonamide binding. The extrapolated increase in K-I over the 0-37 degrees temperature range varied from 4-fold for sulfanilamid e binding to CAI to 14-fold for CF3SO2NH2 binding to CA IV, correspond ing to binding enthalpy values of -7.2 to -11.7 kcal/mol. For CA II an d I, entropy changes associated with sulfonamide binding were in gener al modest and ranged from -5.3 to +4.1 entropy units (eu) for five of the compounds tested. In contrast, ethoxzolamide binding was associate d with a relatively large positive entropy change. Also, the variation e in k(on) and k(off) with temperature were studied for three sulfonam ides binding to CA II. The association rate constants for methazolamid e, benzolamide, and ethoxzolamide binding showed increases of e-fold o r less, whereas dissociation constants increased 3-9-fold over the ran ge of 0-37 degrees. Thus, the temperature effect in increasing K, is i n large part due to a faster rate of sulfonamide dissociation. Apparen t activation parameters at 23 degrees for k(on) were Delta H double da gger = -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(of f), 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 t o -45.7 entropy units.