Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase.
Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs. On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half. Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK. Previous site-directed mutagenesis studies [Li et al. (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem. Biophys. Res. Commun. 290, 670-675] have identified the origins of the citrate and fructose 2,6-bisphosphate sites. Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site. Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433. The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site. It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK. The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.
Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, Illinois 60064, USA. kempr@finchcms.edu
Robert G
RG
Kemp
Dhammika
D
Gunasekera
D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D019343:Citric Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D019343:Citric Acid; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase