Use of a minimum perturbation approach to predict TIM mutant structures

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Triosephosphate isomerase (TIM) is a crucial enzyme in glycolysis.
• Mutations can alter enzyme structure and function, impacting catalytic activity.
• Understanding these structural changes is key to enzyme mechanism studies.

Purpose of the Study:

• To model the conformational structures of yeast TIM single (E165D) and double (S96P/E165D) mutants using computational methods.
• To investigate the structural basis for altered catalytic activity in these mutants.
• To compare the predicted structures with available experimental data (X-ray structures).

Main Methods:

• Minimum perturbation conformational search approach.
• Calculation of minimum energy structures for mutant conformations.
• Analysis of van der Waals and electrostatic interactions, including the role of crystal waters.
• Comparison of calculated structures with experimental X-ray data.

Main Results:

• One calculated minimum energy conformation for the E165D mutant aligned with the X-ray structure, though not the lowest energy conformation.
• Inclusion of active-site water molecules shifted the energy ranking, making the X-ray conformation the lowest energy conformer for E165D.
• All calculated minima for both mutants positioned the Asp side chain to utilize the anti-orbital for proton abstraction, unlike the pseudo-revertant chicken TIM.

Conclusions:

• Predicting the exact orientation of a single amino acid side chain in a mutant protein is complex.
• The orientation of the Asp side chain in yeast TIM mutants may explain the lack of pseudo-revertant activity compared to chicken TIM.
• Computational modeling, especially when including crystal waters, provides valuable insights into enzyme structure-function relationships.