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Glucose 6-phosphate activation of pyruvate kinase from Mycobacterium smegmatis.

ABSTRACT: 1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.

SUBMITTER: Kapoor R

PROVIDER: S-EPMC1162624 | biostudies-other | 1981 Feb

REPOSITORIES: biostudies-other

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Project description:For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

Project description:Many bacteria regulate gene expression in response to phosphate availability using a two-component signal transduction system, the activity of which is controlled by interaction with the Pst phosphate specific transporter and a cytoplasmic protein PhoU. Mycobacterium tuberculosis, the causative agent of tuberculosis, requires its phosphate sensing signal transduction system for virulence and antibiotic tolerance, but the molecular mechanisms of phosphate sensing remain poorly characterized. M. smegmatis serves as a model for studying mycobacterial pathogens including M. tuberculosis. M. smegmatis encodes two proteins with similarity to PhoU, but it was unknown if both proteins participated in signal transduction with the phosphate-responsive SenX3-RegX3 two-component system. We constructed phoU single and double deletion mutants and tested expression of genes in the RegX3 regulon. Only the ?phoU1?phoU2 mutant exhibited constitutive activation of all the RegX3-regulated genes examined, suggesting that M. smegmatis PhoU1 and PhoU2 have overlapping functions in inhibiting activity of the SenX3-RegX3 two-component system when phosphate is readily available. The ?phoU1?phoU2 mutant also exhibited decreased tolerance to several anti-tubercular drugs. However, a complex plasmid swapping strategy was required to generate the ?phoU1?phoU2 mutant, suggesting that either phoU1 or phoU2 is essential for in vitro growth of M. smegmatis. Using whole-genome sequencing, we demonstrated that all five of the ?phoU1?phoU2 mutants we isolated had independent suppressor mutations predicted to disrupt the function of the Pst phosphate transporter, suggesting that in the absence of the PhoU proteins phosphate uptake by the Pst system is toxic. Collectively, our data demonstrate that the two M. smegmatis PhoU orthologs have overlapping functions in both controlling SenX3-RegX3 activity in response to phosphate availability and regulating phosphate transport by the Pst system. Our results suggest that M. smegmatis can serve as a tractable model for further characterization of the molecular mechanism of phosphate sensing in mycobacteria and to screen for compounds that would interfere with signal transduction and thereby increase the efficacy of existing anti-tubercular antibiotics.

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Glucose 6-phosphate activation of pyruvate kinase from Mycobacterium smegmatis.

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