Interaction of bovine carbonic anhydrase with (neutral) aniline, phenol, and methanol

We have investigated the interaction of bovine carbonic anhydrase with neutral aniline, phenol, and methanol molecules. The measurements are of optical spectra and solvent water and methanol proton magnetic relaxation rates of solutions of Co2+-substituted enzyme. We recently proposed a model [Koenig, S. H., Brown, R. D., & Jacob, G. S. (1980) Proceedings of the Symposium on Biophysics and Physiology of Carbon Dioxide, Springer-Verlag, West Berlin and Heidelberg], based on the interaction of enzyme with monovalent anions, that accounts for the pH dependences observed for a wide variety of phenomena, including the apparent pKa for enzymatic activity. We now extend the model to include the observed effects of neutral molecules. Aniline and phenol, though isoelectronic, shift the observed pKa values in opposite directions, and both appear to bind at the aromatic binding site to which sulfonamide inhibitors and aromatic esters are known to bind. The resulting binary complexes behave as altered enzymes, with different values of the pKa for activity, but otherwise are similar to the native enzyme. In terms of our model, aniline and phenol alter the relative affinities of water and anions for the same coordination position of the metal ion at the active site. The effect is opposite in sign for the two nolecules becuase of the differing proton affinities of the NH2 and OH moieties of the phenol ring in each case. By extension, our results indicate that data from experiments using aromatic buffers such as imidazole and lutidine should be analyzed with some care; effects previously attributed to buffer molecules to the aromatic binding site in the active region of the enzyme. The interaction of methanol with carbonic anhydrase is quite different, and very weak. Methanol does displace water at the metal, but to first order there is little, if any, preferential binding of methanol compared to water. Observations by others that alcohols inhibit esterase activity with inhibition constants on the order of 1 M are not attributable to binding of alcohol to enzyme but rather, in our view, result from the increased solubility of aromatic ester substrates in the alcohol-modified solvent.