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Evaluating the Catalytic Importance of a Conserved Glu97 Residue in Triosephosphate Isomerase

Evaluating the Catalytic Importance of a Conserved Glu97 Residue in Triosephosphate Isomerase

Timothy C Chang, Julie H Park, Anh N Colquhoun, Chris B Khoury, Nessa A Seangmany, Jason P Schwans

Biochem Biophys Res Commun. 2018 Oct 28;505(2):492-497.

PMID: 30268499

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9023783-A	Triosephosphate Isomerase from baker's yeast (S. cerevisiae)		9023-78-3	Price

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