Subunit Interactions and Composition of the Fructose 6-Phosphate Catalytic Site and the Fructose 2,6-Bisphosphate Allosteric Site of Mammalian Phosphofructokinase

Abstract

Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541–547

“…In some enzymes the monomeric subunits must dimerize in order to assemble a catalytic center composed of amino acids originating from different polypeptide chains (Qian et al, 2009). In other dimeric enzymes, all the catalytically important amino acids are derived from a single polypeptide chain (Ferreras et al, 2009), and oligomerization only serves to stabilize the structure. Dimerization can also be used to increase the number of binding partners and regulate substrate affinity or selectivity (Marianayagam et al, 2004).…”

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

“…To stimulate PFK1 activity, Fru 2,6-BP binds to a highly conserved site which, in mammals, contains Arg 566, Arg 655 and His 661. These amino acids play a key role in the binding of Fru 2,6-BP to PFK1 (Ferreras et al , 2009). Fru 2,6-BP is produced by the group of enzymes termed 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by the genes PFKFB1-4 .…”

“…A recent study found that UDP-GlcNAc is used to glycosylate Ser 529 on PFK1, resulting in reduced PFK1 activity in a variety of human cancer cells (Yi et al , 2012). Ser 529 is a key residue at the site where Fructose 2,6-BP binds PFK1, forming a hydrogen bond with the 2-phosphate group on Fructose 2,6-BP (Ferreras et al , 2009; Yi et al , 2012). Glucose deprivation induces OGT dependent PFK1 Ser 529 glycosylation, slowing glycolytic flux to allow glucose to be channeled elsewhere in low glucose conditions.…”

Functional Linkage of Adenine Nucleotide Binding Sites in Mammalian Muscle 6-Phosphofructokinase

“…From recent studies, we know that the smallest active enzyme form of mammalian Pfk consists of four subunits but that higher oligomeric complexes exist that are also functional (23,24). Physiologically, AMP and M concentration of ADP promote enzyme activation, and mM concentrations of ADP and ATP promote enzyme inhibition.…”