Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I.

We used a combination of protein engineering and spectroscopic methods to investigate the effect of a long length loop on the conformational stability and activity of chondroitinase ABC I. This study involves manipulation of interactions around Asp(689) as a key residue in the central region of the loop containing residues 681-695 located at C-terminal domain of the enzyme. According to the equilibrium unfolding experiments and considering thermodynamic m value and ΔG(H2O), we found that the folded state of H700N, L701T, and H700N/L701T are more compact relative to the folded state of wild-type protein and they become stabilized upon mutation. However, the compactness and stability of other variants are less than those of wild-type protein. According to enzyme activity measurements, we found that the catalytic efficiency of structurally stabilized variants is decreased, while that of destabilized mutants is improved.