Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2

Abstract

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Keywords: allosteric regulation; amino acids; asparagine; aspartate; cancer metabolism; crystal structure; enzyme kinetics; fluorescence binding; glycolysis; oligomerization; pyruvate kinase; pyruvate kinase muscle isoform 2 (PKM2); valine.

Figure 1.

Effect of Asn, Asp, and Val on PKM2 activity and activation of Val inhibited PKM2 by various effector molecules. The activity assays with Asn, Asp, and Val were performed with 20 nm PKM2 with the PEP concentration varying between 0.1 and 12 mm. For the reactivation assays, the enzyme concentration was 12.5 nm, and the PEP concentration range was 0.1–20 mm. The ADP concentration was kept fixed at 0.8 mm for all experiments. A, varying concentrations of AAs were incubated with PKM2, and kinetic data at 450 μm (Asp and Asn) and 4 mm (Val) concentration were chosen to calculate the catalytic efficiency. The k_cat/K_m increases from 8.2 ± 0.8 × 10³ to 22 ± 3.1 × 10³ mm⁻¹ min⁻¹ in the presence of Asn and from 10.5 ± 2.4 × 10³ to 24.6 ± 1.6 × 10³ mm⁻¹ min⁻¹ with Asp. Val decreases the catalytic efficiency from 8.6 ± 1.4 × 10³ to 1.8 ± 0.2 × 10³ mm⁻¹ min⁻¹. B–E, PKM2 was inhibited with Val (1 or 5 mm), and the activity was resuscitated by adding an increasing amount of Asn, Asp, Ser, and FBP. Kinetic parameters are listed in Table S2. F, displacement of Ser activated PKM2 with increasing concentrations of Val. The catalytic efficiency decreases from 110 ± 17 × 10³ mm⁻¹ min⁻¹ in the presence of 1 mm Ser to 44 ± 4.6 × 10³ mm⁻¹ min⁻¹ in the presence of 1 mm Ser and 2 mm Val. Kinetic parameters are listed in Table S3.

Figure 2.

The presence of Asn, Asp, and Val alters the binding of PKM2 for substrate PEP, but not ADP. A, binding of ADP in the absence (black) and presence of 1 mm Asn (magenta), Asp (salmon), and Val (green), showing that the AAs do not influence the ADP-binding affinity of PKM2. B, PEP-binding affinity of PKM2 in the absence (black) and presence of 1 mm Asn (magenta), Asp (salmon), or Val (green). K_d values were as follows: 10 ± 2 μm (no AA ligand), 0.27 ± 0.0 μm (Asn), 0.29 ± 0.01 μm (Asp), and 81 ± 21 μm (Val). Error bars, S.E.

Figure 3.

Effect of PEP and MgCl₂ on the binding of Asn, Asp, and Val to PKM2. The studies were conducted at a fixed concentration of MgCl₂ (1 mm) and PEP (2 mm) while the concentration of the AA was varied. Shown is binding of PKM2 to Asn in the absence (●) and presence of MgCl₂ (▴)/MgCl₂ + PEP (■) (A), Asp in the absence (●) and presence of MgCl₂ (▴)/MgCl₂ + PEP (■) (B), and Val in the absence (●) and presence of MgCl₂ (▴)/MgCl₂ + PEP (■) (C). The dissociation constants (K_d) of all AAs to PKM2 are listed in Table 1. Error bars, S.E.

Figure 4.

Gel filtration chromatograms depicting changes in the oligomeric state of PKM2 in the presence of allosteric regulators, Asn, Asp, Ser, and Val. The experiments were conducted at fixed concentrations of corresponding AA ligand (10 mm) and enzyme (0.5 and 0.1 mg/ml). PEP, MgCl₂, and FBP concentrations were kept at 5, 10, and 1 mm, respectively. Shown is an overlay of PKM2 with Val (green), Asn (magenta), Asp (orange), and without amino acid (black) (A and D); Val (green), Val + MgCl₂ (brown), Val + MgCl₂ + PEP (red), and Val + FBP (pale blue) (B); and Val (green), Val + Asn (magenta), Val + Asp (orange), and Val + Ser (blue) (C). In A and D, concentrations of PKM2 were at 0.5 and 0.1 mg/ml, respectively. In B and C, MgCl₂, MgCl₂ + PEP, FBP, Ser, Asn, and Asp shift the oligomeric state of Val-inhibited PKM2 from a mixture of tetramer and dimer/monomer equilibrium (green trace) to a near tetramer. The standards (158 and 44 kDa) and theoretical molecular weight of dimer (120 kDa) and monomer (60 kDa) are marked as ticks on the upper axis. As the protein that elutes at ∼13.7 ml corresponds to a calculated molecular weight of 99 kDa, that fraction exists in a dimer/monomer equilibrium.

Figure 5.

Crystal structures of the AA-binding pocket of PKM2 with bound Asn (violet) in chain C (A), Asp (light orange) in chain B (B), and Val (green) in chain C (C). PKM2-AA complex superimposed on PKM2-FBP (cyan, PDB: 1T5A). Dashed lines represent hydrogen bond interactions between the AA and residues involved in binding. Composite omit 2F_o − F_c maps (blue mesh) were generated for Arg-106 and all of the ligands and are contoured at 1σ. For all of the structures, the F_o − F_c omit maps (green mesh) for the AA ligands are contoured at 3σ. Red, oxygen; blue, nitrogen; protein backbone color, carbon.

Figure 6.

Superimposition of the AA-binding site of PKM2 with activators and inhibitors bound. A, PKM2-Asn (PDB: 6V74, chain C, violet), PKM2-Asp (PDB: 6V75, chain B, light orange), and PKM2-Ser (PDB: 4B2D, chain A, pale green). B, PKM2-Val (PDB: 6V76, chain C, green), PKM2-Ala (PDB: 6GG3, chain A, gray), and PKM2-Cys (PDB: 6NU1, chain C, light blue). Spheres, water molecules present in PKM2-Ser and PKM2-Cys. Dashed lines, H-bonds. The side chains of Asn, Asp, and Ser can form H-bonds with neighboring residues in the binding pocket, whereas these interactions are absent in Val, Cys, and Ala. Red, oxygen; blue, nitrogen; protein backbone color, carbon.