Aleixo M. Muise and Kenneth B. Storey
Hexokinase from larvae of the freeze avoiding goldenrod gall moth, Epiblema scudderiana, was purified 20-fold using chromatography on DE52 Sephadex, phosphocellulose, and blue dextran. Final specific activity was 75.8 U/mg and SDS-PAGE gave a molecular weight of 94,000 for the monomer. Arrhenius plot showed a break at 16°C or 12°C in the absence versus presence of 10% v/v glycerol indicating a conformational change in the enzyme at lower temperatures but suggesting a stabilizing effect of glycerol. Comparison of hexokinase kinetic properties at 22°C and 4°C showed higher affinity for both glucose and ATP (Km values were 45-50% lower), as well as for the cofactor Mg2+, at the lower temperature. Furthermore, product inhibition by glucose-6-phosphate and ADP was reduced at 4°C. Glucose levels rise in E. scudderiana as an apparent by-product of high rates of glycogenolysis during glycerol synthesis. The temperature-dependent properties of hexokinase would facilitate the recycling of this glucose back into the pathway of glycerol synthesis and could help to achieve the near stoichiometric conversion of glycogen to glycerol that is seen during cold hardening.