Human Pyruvate Kinase Research Articles

Optimizing 2-furylated imidazole π-bridges for NIR lipid droplet imaging.

Lipid droplets (LDs) are globular biological organelles found in the human body, essential for lipid storage, homeostasis, energy reserve, cellular stress response, membrane biogenesis, and cellular signaling. Dysregulated accumulation of LDs leads to various diseases, including breast and liver cancers. Therefore, the development of diagnostic tools for monitoring LDs using suitable probes for bio-imaging applications is imperative. However, identifying promising probes with near-infrared emission characteristics is still a challenging and intriguing task, requiring extensive exploration of the structure-emission property relationship to design efficient probes for LDs. In this context, we envision the impact of 2-furylated imidazole as a π-bridge and have designed nine LD probes by substituting it with electron-releasing groups like CH3, NH2, NH(CH3), and N(CH3)2 at the 3rd and 4th positions via DFT, TD-DFT, FMO, ESP, NCI, and QTAIM analyses. Our results demonstrate that LDP7 with NH(CH3) at the 3rd position is the most promising molecule, exhibiting the highest emission maxima (772.02 nm) with a lower HOMO-LUMO gap, suggesting its suitability for a range of biomedical applications. An enhancement of ∼200 nm is achieved through tailoring the molecular structure using the designed 2-furylated imidazole-derived π-bridge. ADMET and molecular docking analysis followed by molecular dynamics simulations with the human pyruvate kinase protein reveal these LDPs' bioavailability, binding ability and their stability towards their bio-imaging applications. In summary, our study offers valuable insights to aid researchers in developing and refining various π-linkers for lipid droplet bio-imaging applications.

An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

Structure-Based Design of AG-946, a Pyruvate Kinase Activator.

Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold. AG-946 (1) demonstrated activation of human wild-type PK (half-maximal activation concentration [AC50 ]=0.005 μM) and a panel of mutated PK proteins (K410E [AC50 =0.0043 μM] and R510Q [AC50 =0.0069 μM]), (2) displayed a significantly longer half-time of activation (>150-fold) compared with 6-(3-methoxybenzyl)-4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thieno[2',3':4,5]pyrrolo[2,3-d]pyridazin-5-one, and (3) stabilized PKR R510Q, an unstable mutant PKR enzyme, and preserved its catalytic activity under increasingly denaturing conditions. As a potent, oral, small-molecule allosteric activator of wild-type and mutant PKR, AG-946 was advanced to human clinical trials.

Spectroscopic insights with molecular docking and molecular dynamic simulation studies of anticancer drug 5-fluororouracil targeting human pyruvate kinase m2

This study was conducted to test the efficacy of 5-fluorouracil (5-FU) as an anticancer drug against the human pyruvate kinase isozyme M2 (PKM2) using spectroscopic, molecular docking and molecular dynamic simulation studies. PKM2 fluorescence quenching studies in the presence of 5-FU performed at three different temperatures indicates dynamic quenching processes with single-set of binding (n ≈ 1) profile. The biomolecular quenching constants (k q) and the effective binding constants (K b) obtained are shown to increase with temperature. The calculated enthalpy (ΔH) and entropy changes (ΔS) are estimated to be −118.06 kJ/mol and 146.14 kJ/mol/K respectively, which suggest the possible mode of interaction as electrostatic and hydrogen bonding. Further, these values were used to estimate the free energy changes (ΔG) and that increases with temperature. The negative ΔG values clearly indicates spontaneous binding process that stabilizes the complex formed between 5-FU and PKM2. Far-UV CD spectra of PKM2 in the presence of 5-FU shows decrease in α-helix contents which point towards the destabilization of secondary structure that weakens the biological activity of PKM2. The intrinsic fluorescence study and circular dichroism (CD) spectra showed minor conformational changes of PKM2 in the presence of 5-FU. Additionally, the results obtained from molecular docking and all-atom molecular dynamic simulation study supports the insight of the spectroscopic binding studies, and strengthens the dynamic stability of the complex between 5-FU and PKM2 through H-bonding. This study establishes a paradigm of 5-FU-PKM2 complexation and the efficacy of 5-FU that compromises the biological activity of the targeted PKM2. Communicated by Ramaswamy H. Sarma

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the invitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20ng/μL and 10mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Docking, ADMET and Molecular Dynamics Studies of Plant-Based Phenolics as Tumor Pyruvate Kinase M2 Modulators

The drug discovery is presently threatened by exorbitant costs and is less productive. To challenge the deterioration in the throughput curve, pharmaceutical corporations, and academic scientists are leaning towards plant-based molecules to ascertain new molecules to convert them into pharmacotherapies, and phenolics are the molecules of choice. Natural products, specifically phenolics are undoubtedly explicit sources for development of effective drugs either totally or in a semi-synthetic way and there are numerous examples to support this statement. Molecular docking is chiefly employed to predict novel lead compounds for drug discovery. In this study, phenolics known to act as tumor pyruvate kinase M2 (PKM2) modulators and considered as anticancer agents were subjected to docking studies using Glide and rigorous in silico ADMET studies using the Qikprop suite. In silico investigations were carried out to find out the explicit interactions of the phenolics with PKM2 (PDB IDs 3GR4 and 4B2D) modulators. The docking studies were carried out on phenolics reported as PKM2 modulators. Myricetin was found to be the most suitable anticancer agent for the PKM2 modulation acting on the DASA-binding site with a glide score of -10.746. In addition, the effectiveness of the study was further validated by performing ADMET studies of all the modulators which again established myricetin to be the most favourable one among the selected compounds. It suggested that amongst the “hit list”, myricetin shares the best features to be considered ahead for drug discovery. The results of molecular dynamics simulation studies of myricetin with the DASA-binding pocket of human pyruvate kinase M2 (PDB ID- 3GR4) are strongly in concert with complementary experimental methods discussed above.

Potential allosteric sites captured in glycolytic enzymes via residue-based network models: Phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase

Likelihood of new allosteric sites for glycolytic enzymes, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GADPH) and pyruvate kinase (PK) was evaluated for bacterial, parasitic and human species. Allosteric effect of a ligand binding at a site was revealed on the basis of low-frequency normal modes via Cα-harmonic residue network model. In bacterial PFK, perturbation of the proposed allosteric site outperformed the known allosteric one, producing a high amount of stabilization or reduced dynamics, on all catalytic regions. Another proposed allosteric spot at the dimer interface in parasitic PFK exhibited major stabilization effect on catalytic regions. In parasitic GADPH, the most desired allosteric response was observed upon perturbation of its tunnel region which incorporated key residues for functional regulation. Proposed allosteric site in bacterial PK produced a satisfactory allosteric response on all catalytic regions, whereas in human and parasitic PKs, a partial inhibition was observed. Residue network model based solely on contact topology identified the ‘hub residues’ with high betweenness tracing plausible allosteric communication pathways between distant functional sites. For both bacterial PFK and PK, proposed sites accommodated hub residues twice as much as the known allosteric site. Tunnel region in parasitic GADPH with the strongest allosteric effect among species, incorporated the highest number of hub residues. These results clearly suggest a one-to-one correspondence between the degree of allosteric effect and the number of hub residues in that perturbation site, which increases the likelihood of its allosteric nature.

120-LB: Small Molecule Activator of Pyruvate Kinase Regulates In Vivo Glucose Homeostasis

Background: Nonalcoholic fatty liver disease (NAFLD) is a major factor in the pathogenesis of type-2 diabetes (T2D). Liver pyruvate kinase (PK) inactivating mutations are associated with diabetes in human subjects and PK deficiency associated with severe liver dysfunction and fat accumulation. Aim: To determine whether small molecule activators of PK (PKa) could improve liver metabolism in rats fed a high fat diet. Methods: Sprague Dawley regular chow (RC) or high fat diet (HFD) rats were either treated acutely (IV bolus 11mg/kg/min then primed continuous 0.06 mg/kg/min) or chronically (50 mg/kg/day x 3 weeks). Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Results: PKa dose dependently suppressed glucose production from primary rat hepatocytes. Acute PKa infusion suppressed endogenous glucose production (EGP) by 20% in RC rats. Chronic PKa exposure did not affect body weight in HFD-fed rats. Three-week PKa treatment did not change activity, food intake, whole-body energy expenditure, or body weight in HFD-fed mice. Nevertheless, PKa improved whole-body insulin sensitivity compared with control HFD-fed rats (glucose infusion rate: control 11.8±1.4 vs. PKa 15.3±0.7 mg/(kg-min), P<0.05). Differences in whole-body insulin sensitivity were accounted for by a 15% reduction in EGP and increased insulin-stimulated peripheral glucose uptake (21.2±0.6 vs. 26.4±1.7 mg/(kg-min), P<0.05). Insulin-stimulated hepatic AKT phosphorylation was also significantly improved. Consistent with improving of peripheral insulin sensitivity, liver and muscle triglyceride content (liver: 27.2±2.6 vs. 16.9±1.5 mg/g, P<0.05. muscle: 9.8 ±1.4 vs. 6.7±1.0 mg/g, P<0.05) were improved in PKa-treated rats. In conclusion, activation of pyruvate kinase in rodents protects against HFD-induced insulin resistance. By reducing EGP and insulin resistance, activation of PK may be an exciting potential therapeutic target for T2D. Disclosure A. Abulizi: None. R.L. Cardone: None. S. Siebel: None. C. Kung: Employee; Self; Agios Pharmaceuticals. Stock/Shareholder; Self; Agios Pharmaceuticals. R. Kibbey: None. Funding American Diabetes Association (1-16-IBS-158 to R.K.); National Institutes of Health (R01DK108283-01A1)

Cancer-associatedmutations in human pyruvate kinase M2 impair enzyme activity.

Mammalian pyruvate kinase catalyzes the final step of glycolysis, and its M2 isoform (PKM2) is widely expressed in proliferative tissues. Mutations in PKM2 are found in some human cancers; however, the effects of these mutations on enzyme activity and regulation are unknown. Here, we characterized five cancer-associated PKM2 mutations, occurring at various locations on the enzyme, with respect to substrate kinetics and activation by the allosteric activator fructose-1,6-bisphosphate (FBP). The mutants exhibit reduced maximal velocity, reduced substrate affinity, and/or altered activation by FBP. The kinetic parameters of five additional PKM2 mutants that have been used to study enzyme function or regulation also demonstrate the deleterious effects of mutations on PKM2 function. Our findings indicate that PKM2 is sensitive to many amino acid changes and support the hypothesis that decreased PKM2 activity is selected for in rapidly proliferating cells.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer–tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

Further investigation of inhibitors of MRSA pyruvate kinase: Towards the conception of novel antimicrobial agents

Several novel series of compounds were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as a highly interconnected essential ‘hub’ protein in MRSA, with structural features distinct from the human homologs which makes it a novel antimicrobial target. Several MRSA PK inhibitors (including the hydrazide 1) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to human PK isoforms. Structure–activity relationship (SAR) studies were carried out on the replacement of the hydrazide linker with 3-atoms, 2-atoms and 0-atom linkers and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 μg/mL.

Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form.

Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2(G415R), a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2(G415R) binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2(G415R) is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression.

Inhibitory and Activating Effects of Some Flavonoid Derivatives on Human Pyruvate Kinase Isoenzyme M2.

Pyruvate kinase isoenzyme M2 (PKM2) is expressed excessively in many different cancer types and it plays an important role in the control of glucose metabolism. Thus, it is evaluated as an important target in the development of medication for cancer. The flavonoids comprise a large group of natural products with variable phenolic structures and occur mainly in plants. They are of great interest due to their biological properties. In this study, the effects of various flavonoid derivatives on the PKM2 enzyme activity were analyzed in vitro. The flavonoid derivatives 1 and 2 showed inhibition effect with IC50 values of <60 μM. IC50 values of compounds 3-8 were calculated as 134, 415, 145, 163, 295 μM, and 3.5 mM, respectively. The molecules 9-12 showed an activation effect with values of AC50 of less than 90 μM. The IC50 values of the derivatives 13-17 were calculated as 115, 150, 200, 221, and 275 μM, respectively. The results show that catechin derivatives can be probably used as lead compounds for the design of PKM2 enzyme activators and inhibitors.

Abnormal Levels of PKM2 in Mdcs from Patients with Severe Aplastic

Severe aplastic anemia (SAA) is a hematologic disease characterized by pancytopenia with severe bone marrow failure. Our previous studies have demonstrated that activated myeloid dendritic cells (mDCs) increased in the bone marrow of SAA patients, which might promote Th0 cells to polarize to Th1 cells and cause the subsequent over-function of T lymphocytes and hematopoiesis failure in SAA. The other notable finding in our study is that abnormal expression of pyruvate kinase M2 (PKM2) in mDCs from SAA patients may be one of the reason for mDC hyperfunction. Human pyruvate kinase M2, which catalyzes the last but rate-limiting step of glycolysis, is exclusively expressed in actively proliferated cells ,especially in embryonic and adult dividing/tumor cells. It remains unclear how PKM2 may change in patients with SAA. This study aims at exploring the role of PKM2 in SAA. In this study, the quantitative changes of mDCs in peripheral blood of 15 SAA patients before immunosuppressive therapy (IST) °¢ 13 SAA patients after IST and 26 normal controls were determined by flow cytometry. Results showed that the levels of PKM2 in mDCs were significantly increased in SAA patients(59.1±15.8)%. After IST, the levels of PKM2 in mDCs decreased(42.6±22.1)% (P<0.05).In normal controls group , the level of PKM2 in mDCs was (32.7±20.2)%. These changes reveal that dysregulation of PKM2 expression and activation in mDCs from SAA patients may contribute to hyperfunction of mDCs, thus leading to the imbalance of downstream Th1/Th2 subsets which followed by hematopoiesis failure in SAA. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro effects of some flavonoids and phenolic acids on human pyruvate kinase isoenzyme M2

Pyruvate kinase isoenzyme M2 (PKM2) is one of the most important control point enzyme in glycolysis pathway. Hence, its inhibitors and activators are currently considered as the potential anticancer agents. The effect of 28 polyphenolic compounds on the enzyme activity was investigated in vitro. Among these compounds, neoeriocitrin, (−)-catechin gallate, fisetin, (±)-taxifolin and (−)-epicatechin have the highest inhibition effect with IC50 value within 0.65–1.33 µM range. Myricetin and quercetin 3-β-d-glucoside exhibited the highest activation effect with 0.51 and 1.34 µM AC50 values, respectively. Twelve of the compounds showed inhibition effect within 7–38 µM range of IC50 value. Sinapinic acid and p-coumaric acid showed an activation effect with 26.2 and 22.2 µM AC50 values, respectively. The results propose that the polyphenolics may be the potential PKM2 inhibitors/activators, and they may be used as lead compounds for the synthesis of new inhibitors or activators of this enzyme.

In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

Synthetic analogues of the marine bisindole deoxytopsentin: potent selective inhibitors of MRSA pyruvate kinase.

As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

Exploring putative molecular mechanisms of human pyruvate kinase enzyme deficiency and its role in resistance against Plasmodium falciparum malaria.

Malaria is the third most prevalent cause of global mortality and is an interesting case of evolutionary selection. In response to high frequency of malaria infection, several host genetic factors have been selected, such as Hemoglobin variants, Glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase deficiency. Among these popular host genetic factors, deficiency of pyruvate kinase enzyme is one of the most important factor that provide resistance against malaria. Regulation of this enzyme at the level of transcription is important and several factors may play crucial role in regulation of this enzyme. DNA sequence variation and epigenetic factors modifying transcriptional regulation of gene have been explored in context of several diseases. In the present study, we explored the factors modifying transcription regulation of pyruvate kinase gene with the help of Bioinformatics tools. On the basis of our predictions we hypothesize that any factor that reduces the availability (level) or activity of pyruvate kinase enzyme must play a strong role in resistance to malaria. Thus, factors reducing the activity (loss of function) or level of pyruvate kinase have been selected to provide resistance against malaria primarily in endemic regions.

Mass spectrometric analysis reveals O-methylation of pyruvate kinase from pancreatic cancer cells

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.