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 at the same time, but the binding of one inhibitor increases the Ki for the second by about 50%. While bound as a reversible inhibitor, bromoacetate or iodoacetamide reacts with the basic form of 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. 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. 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. 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. 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.
|Original language||English (US)|
|Number of pages||10|
|Journal||European Journal of Biochemistry|
|State||Published - Sep 1970|
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