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Determining the Catalytic Residues in a Phosphoglucose Isomerase

Presenters Name: 
Katherine Lake
Co Presenters Name: 
Primary Research Mentor: 
Linda Columbus
Secondary Research Mentor: 
Nicole Swope
Time: 
11:00 - 12:15
Time of Presentation: 
2019 - 11:00am to 12:15pm
Session: 
2
Location: 
Newcomb Hall Ballroom
Presentation Type: 
Poster
Presentations Academic Category: 
Science
Grant Program Recipient: 
Not a Recipient
Abstract: 

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P). PGIs are crucial in glycolysis and gluconeogenesis pathways and have been proposed as “moonlighting” proteins in different organisms. In humans, an inactive or inefficient PGI leads to nonspherocytic hemolytic anemia, and PGI elevation is a known marker for tumor metastasis, which highlights the importance of understanding the function of PGIs. The proposed function of Thermotoga maritima PGI (TmPGI), based on structural similarity, was confirmed by kinetic and colorimetric assays in addition to 1H nuclear magnetic resonance (NMR) spectroscopy. The ene-diol mechanism consists of three basic steps: ring opening, isomerization, and ring closure. A glutamate (E281) and a histidine (H310) residue in the active site each have been proposed as the catalytic base that forms the intermediate based on previous PGI crystal structures. To determine which of these residues is the PGI residue critical for base catalysis, alanine mutations, E281A and H310A were formed. The E281A mutant activity and solvent exchange behavior were consistent with wild-type TmPGI at high enzyme concentrations (µM), suggesting that the proposed catalytic glutamate is not critical for PGI function. In addition to the catalytic base activity, TmPGI H310 has been proposed as the residue performing the first ring opening step; thus, the proposed active site histidine residue may be performing both ring-opening and isomerization steps of PGIs that utilize the enediol pathway. Preliminary results with H310A and E281A/H310A will be presented to investigate this hypothesis.