Stereospecificity of the fructose 2,6-bisphosphate site of muscle 6-phosphofructo-1-kinase.
We explored the stereospecificity of the fructose 2,6-bisphosphate site of rabbit muscle 6-phosphofructo-1-kinase by determination of the activation constants (Ka) of several structurally locked analogues of this potent metabolic regulator. Under the assay conditions used, the Ka of fructose 2,6-bisphosphate was 0.12 microM. The most effective synthetic analogues and their Ka's were 2,5-anhydro-D-mannitol 1,6-bisphosphate (2.9 microM), 1,4-butanediol bisphosphate (6.6 microM), hexitol 1,6-bisphosphate (40 microM), and 2,5-anhydro-D-glucitol 1,6-bisphosphate (47 microM). Ten other bisphosphate compounds were much less effective as activators of the enzyme. These findings indicate that, unlike its active site, this allosteric site of 6-phosphofructo-1-kinase does not require the furanose ring. Its basic requirement seems to be a compound with two phosphate groups approximately 9 A apart. Although the free hydroxy groups of the activator do not seem to be essential, their presence enhances appreciably the affinity of the ligand for this regulatory site.
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000378:metabolism*; D006598:Hexosediphosphates / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity
D005635:Fructosediphosphates; D006598:Hexosediphosphates; C027652:fructose 2,6-diphosphate; D010732:Phosphofructokinase-1