| Abstract: | Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B. |
