Investigating enzyme activity is central to our understanding of biological function, and the design of biocatalysts continues to find applications in synthesis. While a role for active site residues can be proposed based on structure and mechanism, our understanding of the catalytic importance for residues surrounding the active site is less well understood. In triosephosphate isomerase (TIM), Glu97 is situated adjacent to the active site and is found in essentially all sequences. Prior studies reported mutation of Glu97 to Asp and Gln in TIM from Plasmodium falciparum (PfTIM) led to a 100- and 4000-fold decrease in activity, respectively, while the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity. To investigate further the question of how mutations in essentially superimposable structures give different effects, we mutated E97 in TIM from Trypanosoma brucei brucei (TbbTIM), Saccharomyces cerevisiae (yTIM), and human (hTIM). The E97D, E97A, and E97Q mutations led to a ∼three-tenfold decrease in activity, a modest effect compared to the 102-103-fold effect in PfTIM. CD and fluorescence studies showed the overall structures for the mutants were essentially unchanged. Structural analysis shows that several residues surrounding E97 differ between PfTIM and TIM from the other organisms, and rearrangements or mispositioning of residues in PfTIM may lead to the different rate effects. The results illustrate the interplay of active site and surrounding residues in affecting catalysis and highlight that understanding of the role of residues surrounding the active site may aid in the incorporation of favorable or avoidance of unfavorable interactions when designing enzymes.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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