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

Abstract

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 20°C and pH 7.5: k_cat of 1.3 × 10⁴ s^{− 1} and k_cat/K_M of 2.5.10⁵ M^{− 1} s^{− 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 (K_Is of 21–78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K_Is around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K_Is in the range of 10–90 μM. It is difficult to explain the inhibitory activity of carbonate (K_I of 10 μM) against hCA III, also considering the fact that this ion has an affinity of 15–73 mM 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 (K_I of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).

Keywords::

• carbonic anhydrase
• isozyme I, II, III
• anions
• sulfamic acid
• sulfamide
• carbonate

Acknowledgements

This work was financed in part by a EU project of the 6^th framework programme (DeZnIT project, contract No. LSHB-CT-2007-037303).