Purification and characterization of the glycolytic enzymes hexokinase and pyruvate kinase from Eurosta solidaginis
Larvae of the insect Eurosta solidaginis spend the winter in galls on goldenrod plants and survive low subzero temperatures by allowing the formation of extracellular ice within their bodies. Different mechanisms are used to allow survival of these extreme conditions: some ensure that the physical integrity of cells is maintained whereas others suppress metabolism so that only basal metabolic functions subsist. Glycolysis, which is at an intersection of several important metabolic pathways, must be subjected to fine regulation to allow the synthesis of the two cryoprotectants of the larvae, glycerol and sorbitol, during the autumn and as well as to facilitate metabolic depression during the winter. In this study, hexokinase (EC 126.96.36.199) and pyruvate kinase (EC 188.8.131.52), two regulatory enzymes of glycolysis, were examined. Hexokinase was partially purified and kinetic studies were performed both at 6°C and 25°C and in the presence and the absence of the cryoprotectants of the insect, glycerol and sorbitol. The data indicate that besides a role in the regulation of glycolysis, hexokinase is probably involved in the control of the synthesis and catabolism of the two polyols. Pyruvate kinase (PK) was purified to near homogeneity and kinetic and structural studies were performed on the purified preparations. Kinetic studies showed that the activity of PK was reduced at cold temperatures and in the presence of the two polyols, with an increase in inhibition by citrate and the loss of activation by fructose-1,6-bisphosphate. Fluorescence and heat denaturation studies underlined the importance of the cryoprotectants, especially sorbitol, for the stabilization of the enzyme.