The Interface of the Glucose-6-Phosphae Dehydrogenase Dimer: Proteolytic Studies

Abstract

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-?-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 determine the interactions taking place between the subunits. G6PDH exits in a rapid equilibrium between its three conformations: monomer, dimer, and tetramer. The dimer conformation of the enzyme has been isolated using our model system from Baker's yeast, Saccharomyces cerevisiae, and visualized with native polyacrylamide gel electrophoresis. Proteolytic degradation of the G6PDH dimer with thrombin and proteinase k has produced two recognizable fragmentation patterns. A change in conformation was predicted to yield a new pattern. This was confirmed with the degradation of the G6PDH tetramer and monomer with both endoproteinases, which produced new fragmentation patterns. The human G6PDH will now replace the model system and be treated with each of its substrates in an attempt to transform its conformation. The enzyme will then be reacted with each endoproteinase. A different fragmentation pattern will confirm that G6PDH changes its conformation in response to substrate binding.