Stereospecificity of the fructose 2,6-bisphosphate site of muscle 6-phosphofructo-1-kinase

Abstract

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.

Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase

“…1(a), Fru(I,6)P2 exerted a half-maximal activation of PFK at 2.2 /M (these concentrations were defined as Ka values) under these conditions. At concentrations above20 JLM, the activity decreased again from its maximal value, as demonstrated previously for L-type PFK[13,38]. All three sugar bisphosphates tested, Fru-…”

“…Among homotetrameric forms of vertebrate PFK consisting of M-type (muscle-type), L-type (liver-type) or F-type (fibroblast-type, also referred to as Por C-type) subunits, the M4 isoenzyme appears to bind hexose bisphosphates other than Fru(2,6)P2 best [2,[17][18][19]. A structural comparison of natural and synthetic bisphosphorylated substances effectively activating M4-type PFK [20,21] indicates that their main similarity is the ability of the two phosphate groups to adopt a distance of about 0.9 nm (9 A). On the other hand, striking differences exist in the structures of the phosphatebearing moieties.…”

“…On the other hand, striking differences exist in the structures of the phosphatebearing moieties. In particular, the presence of a furanose ring seems not to be obligatory, since hexitol 1,6bisphosphate [20], butanediol 1,4-bisphosphate [20], Glc(1,6)P2 and Man(1,6)P2 [10] are also potent activators. However, if an activator is to be active below micromolar concentration, the furanose ring and the trans-orientation of its two free hydroxy groups appear to be necessary [22].…”

The Crystal Structures of Eukaryotic Phosphofructokinases from Baker's Yeast and Rabbit Skeletal Muscle

“…It forms most of the interactions with the protein. We acknowledge the works of Kelley et al 32 and Ishikawa et al, 33 who probed the Fru2,6-P 2 activator site with several analogues of Fru2,6-P 2 . that a tetramer of the mammalian protein resembles a tetramer of ScPFK.…”