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 kcat of 6.7 × 105 s−1 and a kcat/Km 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 (KIs of 0.51–8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs 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.
Highlights
The carbonic anhydrases (CAs, EC 4.2.1.1) are enzymes that effectively catalyze the reaction between CO2 and water, yielding bicarbonate (HCO3 − ) and protons (H+ )
We measured the catalytic activity of the recombinant E. histolytica (EhiCA) [27], comparing its kinetic parameters with those of other such enzymes, belonging to the α-class, such as hCA I and II, (h stands for human isoform)
Like most enzymes belonging to the CA superfamily, EhiCA was inhibited by acetazolamide (AZA, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide): A standard, clinically used sulfonamide CA
Summary
The carbonic anhydrases (CAs, EC 4.2.1.1) are enzymes that effectively catalyze the reaction between CO2 and water, yielding bicarbonate (HCO3 − ) and protons (H+ ). CAs are multifunctional enzymes, which play a central role in different physiological, biochemical, and metabolic processes, such as acid-base homeostasis; respiratory gas exchange; electrolyte secretion; and biosynthesis of urea, glucose, fatty acids, and carbamoyl phosphate They are vital in ionic transport, muscular contraction (in vertebrates), and photosynthesis (in plants and algae). The invasive forms of the infection generally include cyst formation in the liver, which can lead to complications such as pleural effusion, due to the rupture of the cyst [22,26] They disseminate through other extraintestinal organs (e.g., the brain or pericardium) [22,23]. We report the cloning, purification, investigation of the catalytic activity, and the anion inhibition profile of the recombinant enzyme belonging to the β-class, identified in the genome of the pathogenic protozoan E. histolytica, denominated EhiCA
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