Hypercooperativity induced by interface mutations in the phosphofructokinase from Escherichia coli

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein structure-function relationships

Background:

• Phosphofructokinase (PFK) is a key glycolytic enzyme exhibiting allosteric regulation.
• Substrate saturation in E. coli PFK is highly cooperative, suggesting an 'all-or-none' binding mechanism.
• Classical models (concerted and sequential) explain allosteric cooperativity but may not fully capture complex regulatory behaviors.

Purpose of the Study:

• To investigate the role of residue Arg152 at the subunit interface in modulating phosphofructokinase cooperativity.
• To explore the structural and kinetic basis of enhanced allosteric regulation in PFK mutants.
• To determine if mutations can lead to hypercooperativity exceeding classical model predictions.

Main Methods:

• Site-directed mutagenesis of Escherichia coli phosphofructokinase, specifically targeting Arg152 and Glu148.
• Enzyme kinetics assays to measure substrate (fructose-6-phosphate) binding cooperativity (Hill coefficient).
• X-ray crystallography to elucidate the structural basis of interactions at the subunit interface.

Main Results:

• Mutation of Arg152, a residue involved in an inter-subunit ion-pair with Glu148, markedly increased cooperativity (Hill coefficient).
• The Arg152-to-lysine substitution resulted in hypercooperativity exceeding the number of substrate binding sites, suggesting kinetic origins.
• Mutations at Arg152 also enhanced allosteric inhibition cooperativity by phospho-enol-pyruvate.
• Structural analysis revealed electrostatic interactions and potential helix movements across the subunit interface.

Conclusions:

• Allosteric coupling in PFK involves electrostatic interactions and conformational changes propagated across subunit interfaces.
• Kinetic mechanisms contribute significantly to the observed hypercooperativity in enzyme regulation.
• Specific residue mutations can fine-tune enzyme cooperativity beyond predictions of classical allosteric models.