I. The rates of hydrolysis of nitrophenyl esters, catalyzed by bovine carbonic anhydrase, depend on the position of the nitro group and on the size of the acyl residue. The most rapidly hydrolyzed substrate of those investigated is p-nitrophenyl acetate. The catalyzed rates are proportional to both enzyme and ester concentrations. Only in the case of m-nitrophenyl acetate could a value of the Michaelis constant, K , , be estimated, approximately 10 mM. Product inhibition by o-nitrophenol occurs during the enzyme-catalyzed hydrolysis of o-nitrophenyl acetate.2 . The metal specificity of the enzyme seems to be independent of the substrate. Zn(I1) and Co(I1) are about equally effective in restoring esterase activity to the apoenzyme while all other metal ions studied do not activate or have a very small effect.3. The pH-dependence of the esterase activity of both Zn(I1)-and Co(I1)-carbonic anhydrase is very similar to that of the CO, hydration activity, and the basic form of a group with a pK near 7 is required for both reactions. Anionic inhibitors are strongly bound to the enzyme when this group is in its acidic form, but the inhibition is almost abolished a t alkaline pH.4. The inhibitory powers of cyanide and sulfide decrease a t acid as well as at alkaline pH. The results agree with the assumption that, formally, CN-and HS-do not bind to the basic form of the enzyme, while HCN and H,S do not bind to the acidic form.Carbonic anhydrase catalyses the reversible hydration of carbon dioxide with an exceptionally high turnover [l]. I n addition, the enzyme has been shown to act on a number of other carbonyl systems. Pocker and Meany reported the carbonic anhydrasecatalyzed hydration of acetaldehyde [2] and pyridine aldehydes [3,4]. The hydrolysis of certain esters is also catalyzed by the enzyme and this activity has been utilized by several investigators, who used p-nitrophenyl acetate (p-NPA) and other phenolic esters as substrates [5-lo]. Recently, a cyclic sulfonate ester has been shown to be an extraordinarily good substrate [ll] although still surpassed by carbon dioxide by several orders of magnitude.We have studied the kinetics of the hydrolysis of p-NPA and some related substrates to obtain further information about the catalytic mechanism of carbonic anhydrase. The apparent Michaelis constants for these substances are large and could not be determined due to the limited solubility of the substrates in water. Although it may not be feasible to use these Enzyme. Carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1).

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