Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO2 hydration reaction at 20C and pH 7.5: kcat of 1.3104s-1 and kcat/KM of 2.5.105M-1s-1. The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (KIs of 21-78.5mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed KIs around 1mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed KIs in the range of 10-90M. It is difficult to explain the inhibitory activity of carbonate (KI of 10M) against hCA III, also considering the fact that this ion has an affinity of 15-73mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (KI of 0.74mM against hCA III, of 12mM against hCA I and of 85mM against hCA II).