Abstract
In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M(2) enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M(2).
Highlights
Pyruvate and heterotropic cooperative activation with Fru1,6-P2 (8 –10)
We investigated the role of the two natural missense mutations, after site-directed mutagenesis in the PKM2 gene, in the regulation of allosteric properties as well as their effects on the secondary and tertiary structures in comparison with wild-type PKM2 (PK-WT)
The altered metabolic phenotypes associated with tumorigenesis have recently been associated with differential regulation of a glycolytic pathway isozyme, PKM2
Summary
Materials—Culture medium with supplementations (Invitrogen), the QuikChange site-directed mutagenesis kit (Stratagene), restriction enzymes (New England Biolabs), and a sequencing kit (BDT v3.1, Applied Biosystems) were obtained from the indicated manufacturers. 0.5-ml fractions were collected at a flow rate of 0.5 ml/min and assayed for total protein concentration and activity. Intrinsic fluorescence measurements were performed using 50 g/ml wild-type and mutant proteins at 25 °C and pH 7.5 in a Cary Varian Eclipse spectrofluorometer using an excitation wavelength of 295 nm, and the emission intensities were recorded in the range of 310 – 450 nm. The effect of increasing concentrations of Fru-1,6-P2, P-enolpyruvate, and Phe on the wild-type and mutant proteins was investigated. The P-enolpyruvate saturation kinetics were studied over a range of pH to study the effect of pH on Km. Vmax, Km, and Hill number was calculated by fitting the data, Vmaxappϭ EtKcat/͑͑1 ϩ Kana/Hnaϩ ͑Hni/Kini͒ (Eq 6). 0.18 Ϯ 0.04 0.11 Ϯ 0.02 0.168 Ϯ 0.07 a Kinetic parameters were obtained by fitting the data to the Hill equation. b Done at 2.5 mM ADP. c Fru-1,6-P2 was used at a final concentration of 2.5 mM wherever required. d Done at 2.5 mM P-enolpyruvate
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