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Stability for function trade-offs in the enolase superfamily "catalytic module"
Ray A Nagatani1, Ana Gonzalez, Brian K Shoichet
1Department of Biopharmaceutical Science, University of California, San Francisco, 1700 4th Street, Byers Hall, Box 2550, Room 508E, San Francisco, California 94158, USA.
Biochemistry
|May 17, 2007
Summary
Enzyme active sites balance stability and function. In the enolase superfamily, conserved catalytic modules show a stability-function trade-off, with charged residues impacting protein stability and activity.
Area of Science:
- Biochemistry
- Protein Engineering
- Enzyme Catalysis
Background:
- Enzyme catalysis relies on charged and polar active site residues for function, transition state stabilization, and protein stability.
- Previous research suggests a stability-function trade-off, where replacing neutral with charged residues often enhances protein stability.
- The enolase superfamily utilizes a conserved 'catalytic module' for diverse enzymatic reactions, suggesting its evolutionary robustness.
Purpose of the Study:
- To investigate the stability-function trade-off within the conserved catalytic module of the enolase superfamily.
- To determine how individual and combined mutations of catalytic residues in o-succinylbenzoate synthase (OSBS) affect protein stability and function.
Main Methods:
- Site-directed mutagenesis was employed to create alanine substitutions for individual residues, groups, and the entire catalytic module of OSBS.
- Protein stability was measured using techniques to quantify changes in kcal/mol.
- Analysis of additive and nonadditive stability effects in multiple mutants was performed.
Main Results:
- Four out of six individual residue substitutions (K131A, D161A, E190A, D213A) significantly increased protein stability, supporting the stability-activity trade-off.
- The highly conserved E190 residue was the most destabilizing.
- Multiple mutations exhibited nonadditive stability effects, indicating functional interactions within the catalytic module, with observed stabilization being less than predicted additively.
Conclusions:
- The catalytic module of the enolase superfamily demonstrates a stability-function trade-off, consistent with previous observations.
- Interactions among catalytic residues within the module mitigate the expected stability cost of charged residues.
- This system-level organization likely contributes to the catalytic module's repeated use in the evolution of diverse enzyme functions.
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