A distinguishing feature of enzymatic catalysis in comparison to small-molecule catalysts is that enzymes use non-covalent interactions to position substrates and active site residues relative to each other. Although residues implicated in positioning can be readily identified by structural inspection, understanding the catalytic importance of these interactions requires experimental tests. Enzyme active sites often contain networks of interactions for which the functional role cannot be distinguished by the structure alone. In triosephosphate isomerase (TIM), a key enzyme in glycolysis, structural and mutational results suggest an important catalytic role for three active site residues: Lys13, His95, and Glu167. A glutamate residue (Glu97) has been shown to be conserved in almost all species of TIM. Based on X-ray structures, a potential role of Glu97 may be to position Lys13. To investigate the role of Glu97 in TIM catalysis, we mutated Glu97 to Ala, Gln, and Asp in Trypanosoma brucei brucei. The Glu97Ala, Glu97Gln, and Glu97Asp mutations led to an ~10,000-fold, ~40-fold, and ~10-fold reduction in k cat respectively. The similar KM value in Glu97Gln and Glu97Asp mutations relative to wild-type TIM suggests substrate binding is not affected by the mutation. The Glu97Ala mutation led to a slight increased KM relative to wild-type, suggesting that Glu97 may play a role in maintaining the structural stability of TIM. Circular dichroism analysis shows that the E97 mutations do not affect the overall helical structure of TIM. Overall, the results provide evidence for this non-active site residue playing an important role in TIM catalysis.
|Advisor:||Schwans, Jason P.|
|Commitee:||Bhandari, Deepali, Weers, Paul M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 56/01M(E), Masters Abstracts International|
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