Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site [Enzymology]

January 21st, 2014 by Xie, Y., An, J., Yang, G., Wu, G., Zhang, Y., Cui, L., Feng, Y.

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colons, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48°C and a 12°C higher T5015. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix, which are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamic simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.