Bacillus 1,3-1,4-β-glucanases possess a highly conserved disulfide bridge connecting a β-strand with a solventexposed loop lying on top of the extended binding site cleft The contribution of the disulfide bond and of both individual cysteines (Cys61 and Cys90) in the Bacillus licheniformis enzyme to stability and activity has been evaluated by protein engineering methods. Reduction of the disulfide bond has no effect on kinetic parameters, has only a minor effect on the activity-temperature profile at high temperatures, and destabilizes the protein by less than 0.7 kcal/mol as measured by equilibrium urea denatu ration at 37°C. Replacing either of the Cys residues with Ala destabilizes the protein and lowers the specific activity. C90A retains 70% of wild-type (wt) activity (in terms of Vmax), whereas C61A and the double mutant C61A-C90A have 10% of wt Vmax. A larger change in free energy of unfolding is seen by equilibrium urea denaturation for the C61A mutation (loop residue, 3.2 kcal/mol relative to reduced wt) as compared with the C90A mutation (β-strand residue, 1.8 kcal/mol relative to reduced wt), while the double mutant C61A-C90A is ∼0.8 kcal/mol less stable than the single C61A mutant. The effects on stability are interpreted as a result of the change in hydrophobic packing that occurs upon removal of the sulfur atoms in the Cys to Ala mutations © 1995 Oxford University Press.
|Journal||Protein Engineering, Design and Selection|
|Publication status||Published - 1 Sep 1995|
- Cysteine mutants
- Disulfide bond
- Hydrophobic packing
- Site-directed mutagenesis