6-phosphogluconate dehydrogenase from a freeze tolerant insect: Control of the hexose monophosphate shunt and NADPH production during cryprotectant synthesis

Abstract

Kinetic properties of 6-phosphogluconate dehydrogenase (6PGDH) from the freeze-tolerant gall fly larvae, Eurosta solidaginis, are strongly affected by temperature and by the presence of the cryoprotectants glycerol and sorbitol. The enzyme was purified 563-fold with a final sp. act. of 13.5 U/mg protein and a 26% yield. K m values for both substrates 6-phosphogluconate (6PG) and NADP + increased with a drop in assay temperature. The addition of either polyol served to lower these values even in the presence of high salt. 6PGDH appears to be the rate limiting step in the hexose monophosphate shunt (HMS) of this cold-hardy insect providing the reducing power in the form of NADPH needed for the production of sorbitol. The only inhibitor found for 6PGDH was KCl. Inhibition of the enzyme increased with a decrease in temperature. Polyols did not reduce KCl inhibition but they did serve to lower the substrate affinity values for 6PG and NADP + in the presence of high concentrations of salt. An increase in the activation energy determined by an Arrhenius plot showed that there was a conformational change in 6PGDH at temperatures below 5°C. This, along with the inhibition of the enzyme by high salt concentrations, could effectively shut down the biosynthesis of sorbitol at low temperature. Therefore, it is evident that through the regulation of this HMS enzyme E. solidaginis is able to (1) produce the reducing power needed for sorbitol synthesis, and (2) control the production and cessation of sorbitol synthesis.