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Turkish Journal of Biology

DOI

10.3906/biy-1705-37

Abstract

Human carbonic anhydrase II (hCA II) enzyme was firstly expressed using a pET-SUMO expression vector in Escherichia 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 conducted using site-directed mutagenesis. The p-nitrophenyl esterase activity of hCA II variants correlates 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 and determining the resulting affinities towards some benzenesulfonamides. 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 an important role in the folding process and the valine residues are very compatible for the hydrophobic region in the active cavity of this isoenzyme. These mutant proteins will lead to a better understanding of structural functions and drug-based studies in the future.

First Page

935

Last Page

842

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