Structure Influencing Function: Using Glucose-6-Phosphate Dehydrogenase as a Model System for Protein-Protein Interactions
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Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-determining step in the Pentose Phosphate Pathway, a secondary pathway for glucose metabolism in animal cells. G6PDH oxidizes glucose-6-phosphate to 6-phosphoglucono-δ-lactone while it reduces NADP to NADPH. This enzyme is known to be functional when it is in its dimer conformation, and the crystal structure suggests the interface between the two subunits is important to the control function. By observing the conformation of G6PDH as it functions, it is possible to see the interactions between the two subunits. G6PDH from baker's yeast, Saccharomyces cerevisiae , was chosen as a model system for proteolytic degradation. The G6PDH dimer was isolated in solution and visualized using native polyacrylamide gel electrophoresis (PAGE). The enzyme was then reacted with several different endoproteinases. Limited proteolytic degradation is expected to produce a recognizable fragmentation pattern. A change in conformation is predicted to yield a different pattern. This will then confirm that the enzyme does change shape as it begins to function. Once the development of a procedure for the proteolytic degradation of the model system is complete, we will use G6PDH from humans. Coomassie Blue staining will be replaced by the more sensitive technique of Western Blotting to visualize the fragments.