Efficient decreasing of the conformational entropy in a long lengths loop via nature of interactions in chondroitinase ABC I

The conformational entropy has an important contribution to destabilization of the folded state of proteins; it may also affect the activity of enzyme. In this research, we investigated the effect of an interaction network which is established in the middle of a long length loop; located at C-terminal domain of chondroitinase ABC I. The three critical interactions of Asp689 with adjacent residues were changed via site directed mutagenesis and the kinetic parameters of single and combination of double mutants were assessed and compared with that of wild type enzyme. The variants were cloned in pET28a as expression vector. Maximum expression was reached at 27 ˚C, 0.7 mM IPTG in Luria-Bertani medium. After purification of enzymes with Ni-Sepharose column chromatography, the variants were then characterized. It was revealed that kinetic parameters of mutants increase relative to that of wild type enzyme. We concluded that increasing the flexibility of above-mentioned loop and its domain upon deletion of interactions results in improving the activity of enzyme; although it may have destabilizing effect on the structure of enzyme.