Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2
| Autor: | Dhiraj Srivastava, Suparno Nandi, Mishtu Dey |
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| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular Thyroid Hormones Protein Conformation Allosteric regulation PKM2 Crystallography X-Ray Ligands Biochemistry Catalysis Phosphoenolpyruvate Serine 03 medical and health sciences Catalytic Domain Humans Cysteine Amino Acids Enzyme Inhibitors Alanine chemistry.chemical_classification 0303 health sciences Chemistry 030302 biochemistry & molecular biology Membrane Proteins Amino acid Isoenzymes Amino Acid Substitution Tyrosine Mutant Proteins Amino acid binding Carrier Proteins Pyruvate kinase Protein Binding |
| Zdroj: | Biochemistry. 58:3669-3682 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00349 |
| Popis: | Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2-Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2. |
| Databáze: | OpenAIRE |
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