In spite of the advances made in experimental and mutational studies, understanding the importance of remote interactions is crucial for refining the knowledge of enzyme catalysis. In this study, a model system for Glu97 was developed in Homo sapiens triosephosphate isomerase ( hTIM) to investigate the energetic contribution and structural role of this fully conserved glutamate residue in the TIM-catalyzed isomerization reaction. Recombinant human triosephosphate isomerase (hTIM) was altered using site-directed mutagenesis, in which an aspartate, glutamine, or alanine residue was substituted for Glu97. In steady-state kinetics, the E97D variant exhibited the most significant catalytic activity while the E97Q enzyme was the least active. Observing both the forward and reverse directions of the TIM-catalyzed reaction, the results revealed that the enzymatic activity for E97D and E97A TIM was diminished by ~3-fold or less, and the rate was essentially unchanged for the E97D mutation. The E97Q mutant observed a greater rate effect, ~10-fold decrease in kcat and ~20-fold decrease in catalytic efficiency (kcat/ KM). To determine the conformational stability of the WT and mutant hTIM, unfolding of all four enzymes was monitored by circular dichroism, tryptophan and ANS fluorescence spectroscopy. The dimer stability was evaluated by gel-filtration analysis and the mutants showed similar chromatograms compared to that of the WT. The similar behavior observed for the WT and E97D suggests that the Asp mutation has little effect on catalysis, enzyme stability, and the unfolding pathway. On the contrary, the statistical significance observed in the E97Q and E97A mutants suggests that the Gln and Ala mutations affect the stability of the structure and may affect the unfolding pathway. Overall, these point-mutations support the model that remote interactions of Glu97 may have a modest role in catalysis. One explanation is that the direct role of Glu97 may have evolved in the human species and plays a less significant role compared to earlier species in evolution in which Glu97 mutations showed larger rate effects. Possibly, the network of unfavorable interactions is reduced and therefore, the mutational effect of Glu97 is less deleterious in hTIM.
|Advisor:||Schwans, Jason P.|
|Commitee:||Narayanaswami, Vasanthy, Weers, Paul M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 58/04M(E), Masters Abstracts International|
|Keywords:||Conserved residue, Enzyme catalysis, Glutamate 97, Homo sapiens, Remote interactions, Triosephosphate isomerase|
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