Evaluating the Catalytic Role of a Conserved Non‐Active Site Residue in Triosphosphate Isomerase

Abstract

Understanding enzyme function often begins with examination of the active site, but important catalytic roles for residues surrounding the active site have been shown. The active site residues of triosephosphate isomerase (TIM), K12, H95, and E165, have been extensively studied, but the role of the highly conserved E97, situated adjacent to the active site, is not well understood. The proximity of E97 and K12 led to a model where the glutamate positions the active site lysine, and the E97D and E97Q mutations in TIM from Plasmodium falciparum (PfTIM) showed a 100‐ and 4000‐fold decrease in activity, respectively, suggesting an important catalytic role for this residue. In contrast, the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity, challenging E97 as a catalytically critical residue. To investigate further the basis for how the same mutations in essentially superimposable structures give different mutational effects, we studied the effects of mutating E97 in TIM from Trypanosoma brucei brucei (tbbTIM) and Saccharomyces cerevisiae (yTIM). Mutants were constructed and the enzymes were recombinantly expressed and purified. Kinetics, circular dichroism (CD), and fluorescence studies were performed to evaluate the effects of the mutations on enzyme function and structure. A new expression system for generating yTIM mutants was developed to overcome prior challenges in obtaining pure enzymes. Michaelis‐Menten kinetics showed the E97D and E97Q mutants led to an ~three‐fold decrease in activity, a modest effect compared to the 102–103‐fold effect in PfTIM. CD studies showed the mutants contain ɑ‐helical contents of 42% (E97D) and 37% (E97Q), similar to the 42% ɑ‐helical content in wild type. CD and fluorescence chemical denaturation studies showed the midpoint concentration of denaturant was within 0.2 M for the mutants relative to the corresponding wild‐type enzymes, suggesting the mutants have little effect on protein stability. A model for the different functional effects is that variations of the surrounding residues in the TIM isozymes from different organisms allow for such a range of deleterious mutagenic effects. These results highlight the complexity of interactions surrounding the active site. A better understanding of the variations in residues surrounding the active site and their role in positioning residues may aid in the design of enzymes with new functions.Support or Funding InformationThis project was supported by a Single‐Investigator Cottrell College Science Award from the Research Corporation for Science Advancement (22489).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.