TY - JOUR
T1 - Contribution of a disulfide bridge to the stability of 1,3-1,4-p-d-glucan 4-glucanohydrolase from Bacillus licheniformis
AU - Pons, Jaume
AU - Planas, Antoni
AU - Querol, Enrique
N1 - Funding Information:
We are indebted to Teresa Dot for the structural analysis. This work was supported by grant B1O94-0912-C02 from Comisi6n Interministerial de Ciencia y Tecnologfa (CICYT), and by the Centre de Referenda de R + D de Biotecnologia, CIR1T, Generalitat de Catalunya. J.P. gratefully acknowledges a predoctoral fellowship from FPI, Ministerio de Educaci6n y Ciencia, Madrid.
PY - 1995/9
Y1 - 1995/9
N2 - 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
AB - 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
KW - Cysteine mutants
KW - Disulfide bond
KW - Hydrophobic packing
KW - Site-directed mutagenesis
KW - Stability
KW - β-glucanase
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U2 - 10.1093/protein/8.9.939
DO - 10.1093/protein/8.9.939
M3 - Article
C2 - 8746732
AN - SCOPUS:0029560330
SN - 1741-0126
VL - 8
SP - 939
EP - 945
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 9
ER -