NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes

Abstract

Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.

Thermodynamic Parameters for the Association of Fluorinated Benzenesulfonamides with Bovine Carbonic Anhydrase II

“…This observation is in contrast to the results reported by Gerig and co-workers. [19,20] On the basis of binding titrations followed by 19 F NMR spectroscopy, these investigators reported that 2-FBS, 3-FBS, and 4-FBS bound with ligand-to-protein stoichiometries of 2:1 in complexes with HCA I and II, and that the bound ligands were in fast exchange on the NMR timescale (and thus were likely to be in the active site). [19,20] These results are very surprising given the 1:1 stoichiometry that has been demonstrated for almost all other sulfonamide-wild-type CA complexes by a number of biophysical techniques.…”

“…[19,20] On the basis of binding titrations followed by 19 F NMR spectroscopy, these investigators reported that 2-FBS, 3-FBS, and 4-FBS bound with ligand-to-protein stoichiometries of 2:1 in complexes with HCA I and II, and that the bound ligands were in fast exchange on the NMR timescale (and thus were likely to be in the active site). [19,20] These results are very surprising given the 1:1 stoichiometry that has been demonstrated for almost all other sulfonamide-wild-type CA complexes by a number of biophysical techniques. [1,45,46] In search of a reconciliation between these conclusions, we speculated that Gerig and co-workers might have been observing a second, weak binding site (K d obs % 5 mm) that we would not observe under the conditions we used for ITC, because of the greater (50-100-fold) concentration of CA used in NMR spectroscopy (0.5-1 mm) than in ITC (10 mm).…”

“…To address this issue, we attempted to replicate the 19 F NMR titration experiments of Gerig and co-workers. [18][19][20] Our results demonstrate that 4-FBS binds to BCA with a 1:1 stoichiometry (Figure 4).…”

“…[12,[16][17][18][19][20] Gao et al studied the binding of para-substituted benzenesul-A C H T U N G T R E N N U N G fonamides with linear hydrocarbon and fluorocarbon tails (p-H 2 NSO 2 C 6 H 4 CONHA C H T U N G T R E N N U N G (CX 2 ) nÀ1 CX 3 , where X = H or F and n = 1-6) to BCA. [12] As the length of the tails was increased, the values of the dissociation constant K d obs decreased by a factor of five for the hydrocarbon series and 15 for the fluorocarbon series.…”

“…Gerig and co-workers studied the solution-phase binding of fluorinated benzenesulfonamides to HCA I and II by using 19 F NMR spectroscopy. [18][19][20] Their results suggested a binding stoichiometry of 2:1 for 2-fluorobenzenesulfon-A C H T U N G T R E N N U N G amide (2-FBS), 3-fluorobenzenesulfonamide (3-FBS), and 4-fluorobenzenesulfonamide (4-FBS) to wild-type HCA I and II, [19,20] and were in stark contrast to the 1:1 stoichiometry generally observed for the binding of all other arylsulfon-A C H T U N G T R E N N U N G amides to wild-type CA.…”