The anomeric specificity of D-fructose 1,6-bisphosphate activation of yeast pyruvate kinase has been investigated utilizing stopped-flow kinetics and synthetic analogs of D-fructose 1,6-bisphosphate. It has been demonstrated that the immediate product, β-D-fructose 1,6-bisphosphate, of the phosphofructokinase-catalyzed reaction increases the catalytic activity of the enzyme. Although exclusive activation by the a anomer is ruled out by these experiments the possibility that the allosteric site is anomerically nonspecific cannot be excluded owing to experimental limits. 2,5-Anhydromannitol 1,6-bisphosphate, 2,5-anhydroglucitol 1,6-bisphosphate, 1,6-diphosphohexitol, and methyl (α+ β)-D-fructofuranoside 1,6-bisphosphate were tested as activators or inhibitors of the D-fructose 1,6-bisphosphate activation of pyruvate kinase. No activation was observed but inhibition of D-fructose 1,6-bisphosphate activation by 2,5-anhydromannitol 1,6-bisphosphate and 2,5-anhydroglucitol 1,6-bisphosphate was noted. Methyl (α + β)-D-fructofuranoside 1,6-bisphosphate and 1,6-diphosphohexitol also proved to inhibit weakly. The collective data suggest that the allosteric site on yeast pyruvate kinase may be nonspecific with respect to anomeric configuration, but that a C-2 hydroxyl is necessary for activation by D-fructose 1,6-bisphosphate.
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