Abstract
As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1) level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A) as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V) of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.
Highlights
The ATP-dependent enzyme 6-phosphofructo-1-kinase (PFK1, EC 2.7.1.11) catalyzes one of the three irreversible steps of glycolysis, a process that is central to primary metabolism
Citrate binding sites in various organisms Analyses of citrate binding sites on the PFK1 enzymes of various eukaryotic organisms revealed that identical amino acid residues were found at the N-terminus in all examined species (Fig. 1A); more variance was observed among the binding sites at the C-terminus
An important feature of catabolism is the generation of chemically conserved energy in the form of ATP, numerous intermediates of primary metabolism act as precursors for the formation of cellular building blocks that enable cell growth and division
Summary
The ATP-dependent enzyme 6-phosphofructo-1-kinase (PFK1, EC 2.7.1.11) catalyzes one of the three irreversible steps of glycolysis, a process that is central to primary metabolism. It catalyzes the Mg-ATP-dependent phosphorylation of fructose-6phosphate (F6P), resulting in its conversion to fructose 1,6bisphosphate (F1,6P) and the release of Mg-ADP as a byproduct [1]. The allosteric ligand binding sites that developed because of mutations in the C-terminal region enable fine-tuning of the regulatory enzyme in response to elevated levels of specific downstream metabolites
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