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PubMed1979

[Steady state kinetics of pyruvate kinase from muscle].

The dependence of the forward pyruvate kinase reaction on the concentrations of ADP and Mg2+ was studied. It was shown that high total ADP concentrations (2-15 mM) exceeding total Mg2+ concentration in the medium lead to the inhibition of the pyruvate kinase reaction, whereas relatively high Mg2+ concentrations (up to 15 mM) do not cause any inhibition. The kinetics of the reaction can be described in the best way by a scheme incorporating the active PEP . E . Mg2+ . Mg . ADP complex and dead-end complexes containing free ADP. An analysis of the experimental data allows to determine all coefficients of the rate equation and to calculate the values of all kinetic parameters. The values of the constants obtained were used for mathematical simulation of the reaction on the basis of the kinetic scheme given. The mathematical model obtained describes satisfactorily the experimentally determined dependences, which is indicative of the correctness of the model.

Adenosine Diphosphate↗

PubMed2004

Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis.

In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.

Acetates↗

PubMed1982

L-Phenylalanine inhibition of muscle pyruvate kinase.

The allosteric inhibition of M1-type pyruvate kinase from rabbit skeletal muscle by phenylalanine is reciprocally dependent on Mg2+ and phosphoenolpyruvate concentrations. At pH 8, phenylalanine acts as a competitive inhibitor with respect to Mg2+ and phosphoenolpyruvate, and vice versa. Phenylalanine introduces sigmoidicity into the dependence of the reaction velocity on [Mg2+]. In vitro kinetic analysis indicates that phenylalanine inhibition of muscle pyruvate kinase is unlikely to have regulatory significance in vivo.

Allosteric Regulation↗

PubMed1981

Modification of two essential cysteines in rabbit muscle pyruvate kinase by the guanine nucleotide analogue 5'[p-(fluorosulfonyl) benzoyl] guanosine.

Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.

Adenosine Diphosphate↗

PubMed1998

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica.

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50,805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

Actinomycetaceae↗

PubMed1984

Phosphorylation in vivo of red-muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress.

That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.

Aerobiosis↗

PubMed1968

Adipose-tissue pyruvate kinase. Properties and interconversion of two active forms.

1. Extraction of rat epididymal adipose tissue with buffer containing EDTA yields a pyruvate kinase, provisionally called PyK-A, the properties of which resemble in several respects those of the allosteric pyruvate kinase of liver. These properties include co-operative interactions with phosphoenolpyruvate, Mg(2+), K(+), NH(4) (+) and ATP, and sensitivity to activation by fructose 1,6-diphosphate. 2. Extraction in the absence of EDTA yields predominantly a form, PyK-B, that shows both normal Michaelis-Menten kinetics with phosphoenolpyruvate, Mg(2+) and ATP, and co-operative interactions with K(+) and NH(4) (+); this form is insensitive towards fructose 1,6-diphosphate. 3. Both forms yield simple kinetics with ADP, though K(m) values differ in the two systems. In all cases where co-operativity has been demonstrated, Hill-plot n values are between 1.4 and 2.0. 4. The conversion of PyK-A into PyK-B is mediated specifically by fructose 1,6-diphosphate; the reverse reaction is occasioned by EDTA, ATP or citrate. It is thought that a bivalent cation may be involved in this interconversion. 5. Attempts at partial purification have revealed that the enzyme resembles the pyruvate kinase of skeletal muscle, rather than that of liver, in its solubility in ammonium sulphate and elution from DEAE-cellulose. 6. The relevance of these properties in the regulation of pyruvate kinase activity in vivo in adipose tissue is discussed.

Adenosine Triphosphate↗

PubMed1982

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii.

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.

Adenosine Triphosphate↗

PubMed1984

In vivo hormonal control of L-type pyruvate kinase gene expression. Effects of glucagon, cyclic AMP, insulin, cortisone, and thyroid hormones on the dietary induction of mRNAs in the liver.

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.

Adrenalectomy↗

PubMedDate not supplied

Marker for renal cell carcinoma (RCC): the dimeric form of pyruvate kinase type M2 (Tu M2-PK).

OBJECTIVE: The evaluate a potential tumor marker for RCC. Tumor formation is generally linked to an expansion of glycolytic phosphometabolite pools and aerobic glycolyticflux rates. To achieve this, tumor cells generally overexpress a special glycolytic isoenzyme, termed pyruvate kinase type M2. To establish the expansion of phosphometabolite pools pyruvate kinase switches between a tetrameric form with high phosphoenol-pyruvate (PEP) affinity and a dimeric form with a lower PEP affinity. The dimeric form is predominant in all tumors that have been investigated and has been termed TuM2Pk. MATERIALS AND METHODS: We studied: a) the expression of TuM2Pk in RCC by immunohistochemistry using a monoclonal antibody recognizing only the mono- or dimeric form of pyruvate kinase, b) the stability of TuM2Pk in serum by measuring TuM2Pk in 3 patients at different times after taking blood with a two-site immunometric assay, c) the a circadiane rhythm of TuM2Pk in blood by measuring levels every 4 hours in 5 patients, d) TuM2Pk- expression in serum (see 2.) in 5 patients by taking blood from tumor-side vena renalis compared to peripherally blood, e) TuM2Pk (see point 2.) in 40 RCC-patients comparing the results with 39 healthy persons and clinical data of RCC, f) the influence of wound healing to TuM2Pk by measuring serum-levels during a period of more than 12 weeks in 6 patients, g) the individual follow up of 4 patients with RCC stage Robson III for more than 2 years. Comparing TuM2Pk-levels to findings of staging by computed tomography. RESULTS: The isoenzyme TuM2Pk could be demonstrated in RCC and their metastases by immunohistochemistry with a monoclonal antibody specific for pyruvate kinase type M2. In normal kidney cells pyruvate kinase type M2 is not detectable. The stability of TuM2Pk was studied in the serum within 30 minutes. No circadian rhythm was found. Most serum TuM2Pk comes from tumor. Serum evaluation in 39 healthy persons was used to determine normal values, with an upper concentration of 28 U/ml of TuM2Pk (95% percentile of normal healthy persons). Serum evaluation in 40 RCC showed a significant difference to healthy persons and a positive correlation with Robson stage and grading No correlation of TuM2Pk was found with histopathological cell type of tumor diameter. After radical nephrectomy normalization of TuM2Pk level was found within 11 weeks in all localized RCC. Continuously elevated serum levels were seen in metastatic RCC. Individual follow-up seems to be possible. CONCLUSION: Initial discrimination is not possible between localized and metastasized RCC using TuM2PK; however, it is possible to differentiate between benign and malignant renal processes; the specificity under these circumstances is 75%. After successful surgery of localized RCC, an elevated TuM2Pk will be normalized within 11 weeks, and will be remain elevated or will increase again in case of RCC-relapse or metastasis. Thus TuM2Pk would appear to be a useful marker for RCC detection and follow-up.

Antibodies, Monoclonal↗

PubMed2005

Mutagenesis of the active site lysine 221 of the pyruvate kinase from Bacillus stearothermophilus.

Lysine 221 of the pyruvate kinase from Bacillus stearothermophilus was mutated to arginine, leucine, asparatic acid and cysteine. All the mutated enzymes were 10(4) to 10(5) times less active than the wild-type enzyme. The cysteine-free enzyme C9S/C268S, and the enzyme C9S/C268S/K221C, which possessed a unique sulfhydryl group at position 221, were prepared. The former had comparable activity to the wild-type enzyme and the latter was 10(4) times less active. These enzymes were denatured and renatured after aminoethylation. The C9S/C268S/K221C enzyme failed to regain its activity when renatured without aminoethylation; but when it was renatured after aminoethylation, it regained 4.5% of the activity of the C9S/C268S enzyme. This evidence suggests the importance of the Lys221 for the pyruvate kinase activity. The kinetic parameters of the S-aminoethylated C9S/C268S/K221C enzyme suggest that it has decreased affinity for phosphoenolpyruvate.

Amino Acid Substitution↗

PubMed1985

Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets.

Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57 000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvate kinase indicate it is the M2 isoenzyme and that its phosphorylation does not affect its catalytic activity.

Animals↗

PubMed1996

Effects of conserved residues on the regulation of rabbit muscle pyruvate kinase.

A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H. (1994) Biochemistry 33, 6301-6309). Glu233-Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme. Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK. but also that the observed effects are most likely applicable to all isozymic forms of PK.

Allosteric Regulation↗

PubMed1988

Purification of pyruvate kinase from germinating castor bean endosperm.

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.

Journal Article↗

PubMed1976

Modification of the regulatory properties of pyruvate kinase of Neurospora by growth at elevated temperatures.

Pyruvate kinase (EC 2.7.1.40) was isolated from Neurospora crassa mycelium grown at 28 degrees C (PK-28) and at 42 degrees C (PK-42). The regulatory properties, particularly the response towards the allosteric effector fructose 1,6-diphosphate (FDP), was different in the two enzymes. PK-28 showed an activation by FDP but PK-42, under comparable conditions, appeared to be activated by low concentrations of FDP and inhibited by higher ones. For PK-28, complex formation with FDP results in a lowering of the isoelectric point from 6.40 to 5.50, representing the pI of the unliganded enzyme and that of the complex, respectively. In contrast to this, PK-42 exhibits a weak binding to FDP as suggested by a lack of decrease in the isoelectric point on treatment with comparable concentrations of FDP. Studies with quenching of aromatic residue fluorescence of PK-28 and PK-42, following binding of FDP, indicate that although this ligand binds to both types of enzymes the affinity for the two is vastly different. Dissociation constants of 9.3 muM and 0.1 mM were calculated for the binding of FDP to PK-28 and PK-42, respectively. It is concluded that growth at elevated temperatures induced a conformational change in the pyruvate kinase leading to partial desensitization of the allosteric site. The nature of the factor(s) responsible for this change is not understood at present.

Allosteric Regulation↗

PubMed2002

Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency.

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine -248 and 2 single nucleotide substitutions, nucleotide (nt) -324T>A and nt -83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream -324T>A and -248delT mutations were nonfunctional polymorphisms. In contrast, the -83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt -87 and nt -83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

Alleles↗

PubMed1977

Purification and molecular properties of bovine heart pyruvate kinase.

A rapid method is presented for the purification of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from bovine heart. The enzyme obtained is homogeneous by criteria of sodium dodecyl sulphate polyacrylamide electrophoresis and ultracentrifugation and has a specific activity of 260 units/mg. It is a tetramer of 220 000 daltons and S020,w = 10.0 S and possesses no free amino-terminal residue. The amino acid composition is similar to that of the M1 isozyme of rabbit and bovine skeletal muscle. The enzyme is subject to polymerisation to a hexamer of the basic tetramer. The polymeric species has a molecular weight of 1320 000, is promoted at low ionic strength and is undetectable at ionic strength greater than 0.2 by either sedimentation equilibrium or sedimentation velocity measurements. The polymerisation is independent of temperature in the range 5--20degrees C implying that charge interactions rather than apolar interactions are responsible for the process.

Amino Acids↗

PubMed1984

Cyclic AMP-dependent phosphorylation of erythrocyte variant pyruvate kinase.

Cyclic AMP-dependent phosphorylation of a variant erythrocyte pyruvate kinase (PK; EC 2.7.1.40) was studied. This variant PK shows a faster electrophoretic mobility than the normal enzyme. The decreased enzyme activity observed in this variant is associated with a quantitative decrease of enzyme protein. Other parameters are within normal ranges. The partially purified variant PK is phosphorylated with a subsequent increase of k0.5s (phosphoenolpyruvate) similar to the normal control, suggesting that the structural abnormality of the variant enzyme has no influence on the phosphorylation-deactivation mechanism. On the other hand, the variant PK in the erythrocyte was less extensively phosphorylated than PK in normal erythrocytes. This may be the result of abnormal metabolism in the patient's red cells, including increased 2,3-diphosphoglycerate and decreased adenosine triphosphate levels.

Adolescent↗