Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Metabolism

Background:

• Cancer cells rely on glycolysis for energy and proliferation.
• Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme.
• PKM2 activity is regulated by allosteric effectors.

Purpose of the Study:

• To investigate the inhibitory mechanism of cysteine on wild-type PKM2.
• To elucidate the structural basis of cysteine inhibition and its reversal.
• To explore the functional coupling between PKM2's active and amino acid binding sites.

Main Methods:

• Biochemical and kinetic assays to study PKM2 activity.
• Structural determination of the wild-type PKM2-cysteine complex using X-ray crystallography.
• Ligand binding studies employing fluorescence and isothermal titration calorimetry.

Main Results:

• Cysteine inhibits wild-type PKM2 by promoting a shift from tetramer to dimer/monomer.
• Fructose 1,6-bisphosphate reverses cysteine-mediated inhibition by inducing tetramerization.
• Crystal structure reveals cysteine binding at the amino acid site, similar to other amino acids.
• Phosphoenolpyruvate and amino acids exhibit bidirectional binding affinity modulation.

Conclusions:

• Cysteine acts as an allosteric inhibitor of PKM2, affecting its oligomeric state.
• The interaction between the amino acid binding site and the active site is functionally bidirectional.
• Understanding PKM2 regulation by small molecules like cysteine offers insights into cancer metabolism.