On the Interaction of Bovine Cobalt Carbonic Anhydrase with Sulfonamides

Abstract

1.The pHdependence 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,-, appear to compete for one binding site on the enzyme.3. When the sulfanilamide inhibition of the C0,-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 duc 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 x lo4 M-l sec-l a t pH 7.9 and 25". In the presence of CO, , the reaction is slower to an extent which is compatible with a competition for a common binding site. .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.Aromatic and heterocyclic sulfonamides with an unsubstituted sulfonamide group constitute a class of powerful and highly specific inhibitors of carbonic anhydrase. Their physiological effects and their use as therapeutic agents have created a considerable interest in them in the field of molecular pharmacology [1,2]. I n addition, they are useful tools in current work on the elucidation of the structure and function of the enzyme. Whitney et al. [3] [B] that the metalion is required for the strong binding of acetazolamide (5-acetylamido-1,3,4-thiadiazole-2-sulfonamide). Furthermore, t,he sulfonamides have a pronounced effect on the structurc and intensity as well as the optical

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…Many studies used stopped-flow methods with a pH indicator to increase the accuracy of the measure of the rate of hydration. 296,299,300,307,453,454 Pocker and Stone reported that BCA acts as an esterase and measured the hydrolysis of p-NPA by monitoring the absorption of the product, p-nitrophenolate, at 400 nm (ε ≈2.1 × 10 4 M −1 cm −1 ). 455 Using this technique, Pocker measured the kinetics of binding of many ligands by determining the decrease in esterase activity as a function of time.…”

“…307 The investigators used the Co II variant because the UVvisible spectrum of the cobalt changes significantly on binding the sulfonamide, providing a spectroscopic handle. Their results suggest that ligand binding depends on two ionizable groups -one on the inhibitor, and one on the protein.…”

Binding of Human Carbonic Anhydrase to Human Hemoglobin

“…Both forms catalyze the interconversion between carbon dioxide and bicarbonate. The human I form, however, is less active than the I1 form [2,4]. The activity of carbonic anhydrase appears to be controlled by an ionizing group in the active site of the enzyme with a pK, between 5 and 8, a value which is dependent on the ionic strength and the concentration of anionic inhibitors in the solution [2,5].…”

Protein-Based Biosensors with Polarization Transduction

“…The detection limit In some sense this approach has the defects of its virtues: of the metal ions which bind to the active site only Zn strongly promotes the binding of sulfonamides, which makes the assay quite specific. Cobalt(II) is much less effective in this regard (Sven Lindskog & Thorslund, 1968) and generally quenches most fluorophores, making it inaccessible for this type of assay. Other metals which bind to the protein do not promote sulfonamide binding, and cannot be measured by this approach; consequently, it is very specific for zinc.. Another issue is that several of the fluorescent aryl sulfonamides we and others (Chen & Kernohan, 1967) have developed for carbonic anhydrase-based Zn sensing exhibit large increases in their fluorescence lifetimes upon binding to the protein, which largely offset the increases in rotational correlation time, and therefore lead to only modest increases in anisotropy.…”