On the Interaction of Bovine Cobalt Carbonic Anhydrase with Sulfonamides

1 The pH‐dependence of the inhibition of cobalt carbonic anhydrase by three different sulfonamides has been measured employing p ‐nitrophenyl acetate as substrate. The binding of these inhibitors is linked to two ionizing groups. One of these seems to be identical to the group in the enzyme previously shown to control the catalytic activity. The pK of the second group is characteristic for the individual inhibitor and agrees with pK ‐values obtained by titration of the sulfonamides. 2 Sulfanilamide and the anionic inhibitor, NO 3 − , appear to compete for one binding site on the enzyme. 3 When the sulfanilamide inhibition of the CO 2 ‐hydration activity was studied by the stopped‐flow method, some anomalous behavior was observed. From the initial part of the reactions, apparent noncompetitive inhibition was obtained. The rates increased over a short time period, however, to give essentially a competitive pattern. The results can be explained by assuming that the substrate and the inhibitor are competitive, but that equilibration is not immediately achieved due to a comparatively slow rate of dissociation of the inhibitor from the enzyme. 4 The apparent second order rate constant for the formation of the enzyme‐sulfanilamide complex has been estimated as 7 × 10 4 M −1 sec −1 at pH 7.9 and 25°. In the presence of CO 2 , the reaction is slower to an extent which is compatible with a competition for a common binding site. 5 A simple, though not unique, mechanism for the sulfonamide inhibition of bovine cobalt carbonic anhydrase, accounting for both rate and equilibrium data, implies the formation of the enzyme‐inhibitor complex through a direct reaction of the ionized sulfonamide with the inactive, acid form of the enzyme.