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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
Published on: July 14, 2015
Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.
1Department of Biochemistry and Biocenter Oulu, University of Oulu, PO Box 3000, FIN-90014 University of Oulu, Finland.
Protein Engineering, Design & Selection : PEDS
|May 29, 2004
Summary
Mutations in the triosephosphate isomerase (TIM) enzyme's flexible loop 6 affect its catalytic efficiency. Structural changes in loop 6 variants explain altered enzyme activity, impacting biological processes.
Area of Science:
- Enzymology
- Structural Biology
- Protein Engineering
Background:
- The flexible loop 6 of triosephosphate isomerase (TIM) undergoes a conformational switch crucial for its catalytic function.
- Directed evolution generated chicken TIM variants with altered C-terminal hinge tripeptides in loop 6.
Purpose of the Study:
- To enzymatically and structurally characterize chicken TIM variants with mutations in the loop 6 C-terminal hinge.
- To correlate structural adaptations with changes in catalytic efficiency.
Main Methods:
- Directed evolution to generate TIM variants.
- Detailed enzymological characterization (catalytic efficiency measurements).
- X-ray crystallography for structural characterization of variants.
Main Results:
- Six variants (LWA, NPN, YSL, KTK, KVA, NSS) were generated and characterized.
- Variants KVA and NSS showed wild-type catalytic efficiency; LWA, NPN, YSL, and KTK exhibited decreased efficiency.
- Structural analysis revealed significant adaptations in NPN, YSL, and KTK, correlating with reduced catalytic efficiency.
- Bulky side chain replacements at position 3 of the C-hinge favored a closed conformation, impacting activity.
Conclusions:
- Structural adaptations in loop 6 variants directly influence TIM's catalytic efficiency.
- The steric bulk at the C-hinge position 3 is critical for maintaining optimal enzyme function.
- Understanding these structure-activity relationships provides insights into enzyme flexibility and catalysis.
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