Reversible phosphorylation of fructose 1,6-bisphosphatase mediates enzyme role in glycerol metabolism in the freeze-avoiding gall moth Epiblema scudderiana

Abstract

Fructose-1,6-bisphosphatse (FBPase) from larvae of the freeze-avoiding gall moth Epiblema scudderiana occurs in two forms, which are interconverted by reversible phosphorylation and separable by CM-cellulose column chromatography. The phosphoenzyme has properties that would make it the more active form in vivo. Compared with the dephosphorylated form, the phosphoenzyme had three-fold lower values for K m fructose-1,6-bisphosphate and K a Mg 2+ and lower sensitivities to allosteric inhibitors (I 50 values for fructose-2,6-bisphosphate and AMP were 50% and 10-fold higher, respectively). The proportions of the two enzyme forms in the larvae changed with the seasons and with acclimation to warm (15°C) vs cold (4°C) temperatures. The phosphorylated enzyme predominated (70% of total activity) in early autumn and during the spring, as well as in warm acclimated larvae, all situations where gluconeogenesis via FBPase would be favoured. During the autumn cold-hardening period when the larvae are actively synthesizing the antifreeze, glycerol, the ratio of the two enzyme forms changed to about 50:50. This, plus allosteric inhibition and low temperature effects on enzyme kinetics, would effectively suppress FBPase activity and prevent futile recycling of glycerol carbon back into glycogen during the winter months when the 2 M pool of polyol must be sustained for antifreeze protection. Acclimation studies suggested that low temperature itself might be the signal that triggers enzyme dephosphorylation, and this could integrate control over FBPase with the well-known phosphorylation-mediated activation of glycogen phosphorylase by low temperature in cold-hardy insects.