FRUCTOSE-1,6-BISPHOSPHATASE FROM A COLD-HARDY INSECT - CONTROL OF CRYOPROTECTANT GLYCEROL CATABOLISM

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 +/- 6.5 kD and a subunit molecular weight of 37.6 +/- 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer al 22 degrees C and rose to 7.31 at 5 degrees C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 +/- 4.1 kl/mol(-1). K-m values for FBPase decreased from 4.7 +/- 0.34 mu M al 22 degrees C to 1.3 +/- 0.05 mu M at 5 degrees C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2, 6P(2)), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCl. inhibition by AMP and F2, 6P(2) increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FB Pase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I-50 values for AMP and F2, 6P(2) increase at 22 degrees C (compared with 5 degrees C), high glycerol levels override F2, 6P(2) inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. (C) 1995 Wiley-Liss, Inc.