Inhibition and Modification of Human Carbonic Anhydrase B with Bromoacetate and Iodoacetamide

Abstract

1. Bromoacetate and iodoacetamide reversibly inhibit human erythrocyte carbonic anhydrase B. The binding of these inhibitors is competitive with that of the other inhibitors, chloride and sulfanilamide. However, bromoacetate and iodoacetamide are only partially competitive with each other. That is, both can bind a t the same time, but the binding of one inhibitor increases the Ki for the second by about 50°/,.2. While bound as a reversible inhibitor, bromoacetate or iodoacetamide reacts with the basic form o f a specific histidine residue in the enzyme. The half-times of reaction are 0.44 h and 2.7 h, respectively. The pH-dependence of reaction shows that the pK of this histidine is 5.6 when bromoacetate is bound as a reversible inhibitor, and the pK when iodoacetamide is bound is below 5.0. 3.Reaction with bromoacetate introduces a carboxymethyl group in the region of the active center. This group perturbs the active site of the enzyme. This specific carboxymethylation causes about a five-fold decrease in esterase activity and a shift in the midpoint of the pH-dependence curve of activity from about pH 7 . 3 to 9.2. The affinity for several inhibitors is decreased. 4.Results of titration of the carboxymethyl enzyme with acetazolamide are consistent with 1.0 binding sites per enzyme molecule. This is the same number of sites as for native enzyme, but the Ki is higher with modified enzyme.5 . Studies of the visible spectrum of cobaltous carbonic anhydrase B have revealed two pH-dependent transitions. The first occurs below pH 7 and produces relatively small changes in the spectrum. Introduction of the carboxymethyl group has little effect on the first transition, but shifts the midpoint of the second transition from pH 7.1 to 9.2.6. Several lines of evidence indicate that the carboxymethyl group in the modified enzyme occupies a position different from the bromoacetate ion in the reversible complex.Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide. (For a review of this subject, see Maren [I].) However, the enzyme will also catalyze other reactions, such as the dehydration of aldehydes [a] and the hydrolysis of esters [3-51. The latter activity is very useful because of the simplicity of the assay.Carbonic anhydrase contains one mole of Zn2f which is essential for activity. The metal can be removed and activity regenerated by addition of Zn2+ or Co2+. The cobaltous enzyme has a visible spectrum which is sensitive to changes in pH and to the addition of inhibitors [6--81. Reaction with these reagents introduces a perturbing group in the region of the active center. Reaction causes complete inactivation in the case of the substituted sulfonamide and over 80°/, inactivation with the other reagents. These reagents react with human carbonic anhydrase A in the same way as with

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