6-PHOSPHOGLUCONATE DEHYDROGENASE FROM A FREEZE-TOLERANT INSECT - CONTROL OF THE HEXOSE-MONOPHOSPHATE SHUNT AND NADPH PRODUCTION DURING CRYOPROTECTANT SYNTHESIS

Kinetic properties of 6-phosphogluconate dehydrogenase (6PGDH) from th e freeze-tolerant gall fly larvae, Eurosta solidaginis, are strongly a ffected by temperature and by the presence of the cryoprotectants glyc erol and sorbitol. The enzyme was purified 563-fold with a final sp. a ct. of 13.5 U/mg protein and a 26% yield. K-m values for both substrat es 6-phosphogluconate (6PG) and NADP(+) increased with a drop in assay temperature. The addition of either polyol served to lower these valu es even in the presence of high salt. 6PGDH appears to be the rate lim iting step in the hexose monophosphate shunt (HMS) of this cold-hardy insect providing the reducing power in the form of NADPH needed for th e production of sorbitol. The only inhibitor found for 6PGDH was KCI. Inhibition of the enzyme increased with a decrease in temperature. Pol yols did not reduce KCI inhibition but they did serve to lower the sub strate affinity values for 6PG and NADP(+) in the presence of high con centrations of salt. An increase in the activation energy determined b y an Arrhenius plot showed that there was a conformational change in 6 PGDH at temperatures below 5 degrees C, This, along with the inhibitio n of the enzyme by high salt concentrations, could effectively shut do wn the biosynthesis of sorbitol at low temperature. Therefore, it is e vident 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.