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Ground state destabilization from a positioned general base in the ketosteroid isomerase active site
Eliza A Ruben1, Jason P Schwans, Matthew Sonnett
1Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Biochemistry
|January 15, 2013
Summary
Bacterial ketosteroid isomerase (KSI) uses a charged Asp general base, which is destabilizing in the ground state. Mutations relieving this charge enhance binding, indicating ground state destabilization contributes to KSI catalysis.
Area of Science:
- Biochemistry
- Enzyme catalysis
- Protein engineering
Background:
- Bacterial ketosteroid isomerase (KSI) catalyzes a key step in steroid metabolism.
- The enzyme's active site features an anionic Asp general base crucial for catalysis.
- Understanding the role of electrostatic and hydrophobic interactions in enzyme active sites is vital.
Purpose of the Study:
- To quantify ground state destabilization caused by the anionic Asp general base in KSI.
- To investigate the impact of charge neutralization on substrate binding affinity.
- To explore the contribution of ground state destabilization to the overall catalytic efficiency of KSI.
Main Methods:
- Site-directed mutagenesis of the Asp38 general base to uncharged residues (Asn, Ala) in bacterial KSI.
- Comparison of binding affinities of ground state analogues with wild-type and mutant enzymes.
- Structural analysis to understand residue interactions within the active site.
Main Results:
- Mutations of Asp38 to Asn or Ala significantly increased analogue binding affinity (1-2 orders of magnitude).
- This suggests that the proximity of the anionic Asp and the hydrophobic steroid is destabilizing in the ground state.
- Mutations at Pro39, adjacent to Asp38, modulated the binding effects, supporting a model of active site reorientation.
Conclusions:
- Ground state destabilization by the charged Asp38 general base makes a modest but significant contribution to KSI catalysis.
- Enzymes evolve for catalytic function, not solely for maximal ground state binding.
- This mechanism of ground state destabilization may be prevalent in enzymes utilizing anionic general bases to deprotonate carbon acids.
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