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Dissertation/Thesis Abstract

Evaluating the Structural Role of a Conserved Glutamate Residue in Triosephosphate Isomerase from Trypanosoma brucei brucei
by Khoury, Chris B., M.S., California State University, Long Beach, 2017, 56; 10604764
Abstract (Summary)

It is well known that enzymes differ from small-molecule catalysts by use of non-covalent interactions to position active site residues, but our understanding of the relative importance of residues in this positioning is limited. Active site residues participate directly in covalent and non-covalent interactions with substrates, but second shell residues may also contribute indirectly to catalysis through positioning and structuring. In triosephosphate isomerase (TIM), a key glycolytic enzyme, a highly conserved glutamate residue at position 97 has been suggested to be important for catalysis and may be important for positioning a key active site lysine residue (K13). In Trypanosoma brucei brucei (TBB), a kinetoplastid which causes African sleeping sickness, Glu97 has been shown to be catalytically important. Mutations of Glu97 to Gln, Asp and Ala have been shown to lead to approximate 24-, 18-, and 6280-fold kcat decreases, respectively. Whereas this glutamate residue is involved in the catalysis of the enzyme, the nature of its involvement in the structure of the enzyme is unclear. To evaluate the role of this residue in the structure of the enzyme, we performed structural and denaturation evaluations using intrinsic and ANS fluorescence. Our results suggested that the Glu97Asp and Glu97Gln mutations did not significantly perturb the structure of the enzyme compared with the wild-type, but may have slight structural effects, based on spectral center of mass and λmax values. Denaturation evaluations suggested that the Glu97Asp and Glu97Gln did not significantly destabilize the enzyme based on [GdnHCl]1/2. The effect of the Glu97Ala mutation, however, was less clear. Overall, our results suggested that although Glu97 is important to catalysis, Glu97Asp and Glu97Gln mutants do not appear to significantly perturb structure, but may have slight effects. Future directions include continued investigation of Glu97Ala, and evaluation of the structural effects of double mutants at the Glu97 and Lys13 positions.

Indexing (document details)
Advisor: Schwans, Jason P.
Commitee: Bhandari, Deepali, Weers, Paul M.M.
School: California State University, Long Beach
Department: Chemistry and Biochemistry
School Location: United States -- California
Source: MAI 57/01M(E), Masters Abstracts International
Subjects: Biochemistry
Keywords: Conserved, Enzymology, Glutamate, Structure, Triosephosphate isomerase
Publication Number: 10604764
ISBN: 978-0-355-28023-4
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