Nitrogen-15 nuclear magnetic resonance study of benzenesulfonamide and cyanate binding to carbonic anhydrase

The binding of inhibitors, cyanate and benzenesulfonamide, the active-site zinc of human carbonic anhydrase was studied by ^(15)N nuclear magnetic resonance . The cyanate nitrogen resonance moved 34 ppm on binding to the enzyme. The shielding is comparable to that reported for a zinc-isocyanate complex and strongly suggests complexation of cyanate to zinc through nitrogen. The proton-coupled ^(15)N resonance of the enzyme-bound benzenesulfonamide was a doublet. Hence, benzenesulfonamide is bound as C_6H_5SO_2NH-. The proton-decoupled ^(15)N resonance of the bound benzenesulfonamide was observed 17 ppm upfield of that of free enzenesulfonamide anion. A model ligand, 2-aminobenzenesulfonamide anion, undergoes binding of zinc through the sulfonamide nitrogen which results an 11.8 ppm shielding of the ^(l5)N resonance. In contrast, N-( 2-aminophenyl)benzenesulfonamide, which is reported to bind zinc through an oxygen, has its sulfonamide nitrogen deshielded by 4.3 and 1.2 ppm on complexation of zinc to the neutral and anionic ligands, respectively. Thus, coordination to the nitrogen causes shielding and to the oxygen deshielding of the sulfonamide resonance. The observed shielding of the enzyme-bound sulfonamide resonance strongly suggests that benzenesulfonamide binds primarily to zinc through the sulfonamide nitrogen. The implications of these results for the high affinity of association of the inhibitor are discussed.