Determining Conformational Changes of G6PD Via Limited Proteolysis
Mentor:Chris Craney, Professor of Chemistry, Occidental College
Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step in the pentose phosphate pathway. This enzyme is functional as dimer and provides the majority of a cell’s NADPH to be used in reductive processes. NADP is known to stabilize the dimer interface of G6PD. Different substrate analogues were added to G6PD to determine the occurrence of hypothetical conformational changes induced by these substrates. Using different substrate analogs to bring about conformational changes would allow a better understanding of the interactions between the subunits that grant the enzyme its activity. G6PD isolated from bakers yeast (Saccharomyces cerevisiae) served as a basis for later experimentation with human G6PD. A number of limited proteolytic analyses were performed by saturating G6PD with substrate, substrate analogues, and inhibitors–NADP, NAD, thioNAD, G6P, ATP, and DHEA– which were subsequently digested with subtilisin, a selective endoprotease. The resulting digestion was then analyzed via SDS-PAGE, producing characteristic fragmentation for each digestion. Comparisons of SDS-PAGE results indicate that only NADP induces a conformational change to stabilize G6PD evinced by a slower degradation of the main G6PD band compared to the G6PD control digest. G6P, however, did not produce any difference in fragmentation, suggesting NADP binding first is critical to G6PD catalytic function. Human G6PD digests using NADP, G6P, and DHEA suggests that NADP and DHEA confer stability to the human variant of G6PD again evinced by decreasing the rate of digestion of the main G6PD band by subtilisin.