Abstract
Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the pentose phosphate pathway, which oxidizes glucose to 6-Phosphoglucono-δ-lactone and reduces NADP to NADPH. G6PDH exists in an equilibrium between its monomer, dimer, and tetramer state.The catalytically active enzyme functions in its dimer conformation. G6PDH has a structural NADP site in proximity to the dimer interface. Functional G6PDH requires the binding of both glucose-6-phosphate (G6P) and NADP, which are believed to hold protective effects in stabilizing G6PDH into its catalytically active conformation. By using limited proteolytic degradation to compare the fragmentation band patterns of substrate-saturated G6PDH and substrate analogue-saturated G6PDH with native G6PDH, it will be possible to observe structural changes around the enzyme?s dimer interface that might lead to changes in it stability. The G6PDH dimer from baker?s yeast, Saccharomyces cerevisiae, will be used as a model system in preparation for work with human G6PDH. In order to spur G6PDH into its catalytic form without beginning catalyst, only NADP will saturate the enzyme. When highly selective proteinases are introduced, G6PDH will be cut into a specific set of fragments, visualized by SDS PAGE. If successful, the experiment would confirm the protective effects of NADP on G6PDH and that G6PDH does undergo a structural change to become functional, which have only been theorized. Subsequently, the fragments could be isolated for sequence analysis that might identify the dimer interface. This could lead to a greater understanding of the dimer interface, the site of mutation occurrence, which causes enzyme deficiency.
