A comprehensive understanding of the design of proteins puts heavy emphasis on certain key residues. These key residues can often be identified by the level of conservation in nature, which acts as a reliable witness mark in order to study the pressures that select for residues which play a critical role in the function of the protein. In the case of triosephosphate isomerase (TIM), the fifth enzyme in glycolysis, the second-shell residue Glu97 has been found to be fully conserved across all known TIM sequences. Its proximity to the active site as well as several previous studies has pointed to a possible direct role in catalysis. However, the present study shows when Glu97 is mutated to Ala, Gln, and Asp in Trypanosoma brucei brucei (tbb) that the resulting effects on kcat are small. Previous results from other studies that have observed larger mutational effects may be due to nearby non-conserved residues that are specific to the TIM homolog in which these studies are performed. The structural studies detailed here suggest that instead, Glu97 is involved in the structural stability of the enzyme, as well as participating in dimer formation. Size-exclusion chromatography analysis suggests that several tbbTIM mutants may in fact be monomeric.
|Advisor:||Schwans, Jason P.|
|Commitee:||Narayanaswami, Vasanthy, Weers, Paul M. M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 58/05M(E), Masters Abstracts International|
|Keywords:||Enzymology, Protein design, Triosephosphate isomerase|
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