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Biochemistry. 1999 Dec 7;38(49):16407-12.

Identification of allosteric sites in rabbit phosphofructo-1-kinase.

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  • 1Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, Illinois 60064, USA.


Earlier studies indicated an evolutionary relationship between bacterial and mammalian phosphofructo-1-kinases (PFKs) that suggests duplication, tandem fusion, and divergence of catalytic and effector binding sites of a prokaryotic ancestor to yield in eukaryotes a total of six organic ligand binding sites. The identities of residues involved in the four binding sites for allosteric ligands in mammalian PFK have been inferred from this assumed relationship. In the current study of the C isozyme of rabbit PFK, two arginine residues that can be aligned with important residues in the catalytic and allosteric binding sites of bacterial PFK and that are conserved in all eukaryotic PFKs were mutated. Arg-48 was suggested previously to be part of either the ATP inhibitory or the adenine nucleotide activating site. However, the mutant enzyme showed only slightly less sensitivity to ATP inhibition and was fully activatable by adenine nucleotides. On the other hand, sensitivity to citrate and 3-phosphoglycerate inhibition was lost, indicating an important role for Arg-48 in the binding of these allosteric effectors. Mutation of Arg-481, homologous to an active site residue in bacterial PFK, prevented binding and allosteric activation by fructose 2,6-bisphosphate. A new relationship between the allosteric sites of mammalian PFK and bacterial PFK is proposed.

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