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

We report the cloning and catalytic activity of a -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 x 10(5) s(-1) and a k(cat)/K-m of 8.9 x 10(7) M-1 x 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(I)s of 0.51-8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (K(I)s in the range of 28-86 M). Since -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.