Cloning, purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

Abstract

<p>A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO<sub>2</sub> + H<sub>2</sub>O ⇋ HCO<sub>3</sub><sup>−</sup> + H<sup>+</sup> with a k<sub>cat</sub> of 1.1 × 10<sup>5</sup> s<sup>−1</sup> and a k<sub>cat</sub>/K<sub>m</sub> of 7.58 × 10<sup>6</sup> M<sup>−1</sup> × s<sup>−1</sup>. This activity was inhibited by acetazolamide (K<sub>I</sub> of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (K<sub>I</sub> of 81 µM), N,N-diethyldithiocarbamate (K<sub>I</sub> of 67 µM) and sulphamic acid (K<sub>I</sub> of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.</p>