Cloning, Characterization and Anion Inhibition Studies of a β-Carbonic Anhydrase from the Pathogenic Protozoan Entamoeba histolytica

Abstract

We report the cloning and catalytic activity of a β-carbonic anhydrase (CA, EC 4.2.1.1), isolated from the pathogenic protozoan Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO₂ hydration reaction, with a k_cat of 6.7 × 10⁵ s^-1 and a k_cat/K_m of 8.9 × 10⁷ M^-1 × s^-1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (K_Is of 0.51⁻8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (K_Is in the range of 28⁻86 μM). Since β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.

Keywords: Entamoeba histolytica; anions; carbonic anhydrase; inhibitor; metalloenzymes; protozoan.

Figure 1

SDS PAGE of the β-CA from E. histolytica (EhiCA) showing a 21/25 kDa doublet polypeptide and additional polypeptides of about 50 and 75 kDa (arrows). Left lane: Standard Mw markers. The dimer and the trimer of EhiCA are also seen (arrows), as reported for other β-CAs cloned and purified earlier [11,12,13,14,15].

Figure 2

Multi-alignment of the amino acid sequences of the β-CAs from M. tuberculosis (isoform MTCA1_MYCTU), Synechocystis sp. (SYNY3), V. cholerae (VIBCL), H. influenzae (HAEIN), E. coli (ECOLI), S. typhimurium (SALTY), E. histolytica (ENTHI), and M. tuberculosis (isoform MTCA2_ MYCTU) [11,27,28,29,30]. Conserved amino acids depicted by an asterisk (*), semiconserved ones by (.) or (:).