Cloning, expression in E. coli, characterization of human carbonic anhydrase ii (hCA II) enzyme, production of mutant hCA II genes by site directed mutagenesis and investigation of affinity of mutant proteins to inhibitors

Human carbonic anhydrase II (hCA II) enzyme was firstly expressed using pET SUMO expression vector in E. coli and the recombinant enzyme was purified using nickel (Ni2+) affinity chromatography. The substitutions of Trp 209 with four amino acid (Val, Leu, Ile, and Pro) in the hydrophobic pocket of hCA II were made using site-directed mutagenesis. The p-nitrophenyl esterase activity of hCA II variants correlate with the hydrophobicity and size of residue, suggesting that the hydrophobic character of this residue is important for catalysis. The Trp 209 was forecast as an important residue and was exposed to computational mutagenesis. This forecast was confirmed experimentally by producing hCA II mutants (W209V, W209L, W209I, W209P) and determining the resulting affinities towards some benzenesulfonamides. Affinity for benzenesulfonamides was reduced in the four mutants compared to wild type (wt). The W209V mutant showed similar specific activity values with the wt enzyme, whereas the W209P, W209L and W209I mutants were less active around 60%, 30%, 20% in p-nitrophenyl esterase activity compared to the wt, respectively. These mutations in the hydrophobic pocket of the enzyme active site decreased the protein expression of hCA II in E. coli, causing the formation of insoluble protein aggregates in many cases. Our findings demonstrated that the Trp 209 in hCA II plays important role the folding process and the valine residues are very compatible for hydrophobic region in active cavity of this isoenzyme. These mutant proteins will lead to the understanding of structural functions of enzyme and drug-based studies in the future.