Fructose-1, 6-bisphosphatase of an extreme thermophile.

Abstract

D-Fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] from Flavobacterium thermophilum HB8, an extremely thermophilic bacterium, was partially purified. The preparation was free from non-specific phosphatase activity and its specific activity was 8 times that of the crude extract. The enzyme specifically hydrolyzed the C-l phosphate ester bond of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The enzyme was similar to the E. coli fructose 1,6-bisphosphatase as regards optimum pH (8.1), requirement for divalent ions, and Km for D-fructose 1,6-bisphosphate (2×10−5M). The substrate, D-fructose 1,6-bisphosphate, was inhibitory to the enzyme at high concentrations. The enzyme was activated by phosphoenolpyruvate, which released the substrate inhibition. AMP inhibited the enzyme activity, emphasizing the substrate inhibition. Inactivation by AMP was recovered to some extent by the addition of phosphoenolpyruvate. The enzyme was heat resistant and both the catalytic and allosteric properties of the enzyme remained unchanged after treatment at high temperatures, for instance, at 70°C for 1 hr. The role of phosphoenolpyruvate in the regulation of glycolysis-gluco-neogenesis in the bacterium is discussed.