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PubMed1977

[Significance of kinetic abnormalities of erythrocyte pyruvate kinase in congenital deficiencies].

Kinetic studies of semi-purified pyruvate kinase were performed from the erythrocytes of 22 subjects with congenital non-spherocytic anemia with pyruvate kinase deficiency and 25 heterozygous relatives. The study of enzyme affinity for PEP with and without ligands fructose-diphosphate (FDP) and ATP allowed to separate three groups of patients: --in eight patients and their eleven heterozygous relatives, the PK kinetic characteristics were similar to those of controls. Allosteric properties of the enzyme were normal; --in three patients, PK was in an activated form, non sensitive to FDP activation and to ATP inhibition in the experimental conditions. The deficient enzyme seemed to be present in the "R" form; --in eleven patients affinity for PEP was decreased; the enzyme was in each case activated by FDP; however very high FDP concentrations did not allow to obtain a complete activation and an n Hill of 1. ATP inhibition was more important than in controls. Similar results were obtained in some heterozygous relatives. The pyruvate kineases of these patients seemed to be fixed in "T" form. Whatever may be the molecular abnormalities responsible for the PK deficiency, they seem to lead in many cases to a change in the enzyme allosteric properties.

Adenosine Triphosphate↗
PubMed2002

Pyruvate kinase type M2: a crossroad in the tumor metabolome.

Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Animals↗
PubMed1997

The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

Amino Acid Sequence↗
PubMed1984

Specific inhibition of L-type pyruvate kinase by the triazine dye Procion Blue MX-R.

Incubation of the triazine dye Procion Blue MX-R with L- and M-type pyruvate kinase resulted in rapid time- and dye-concentration-dependent loss of activity. L-type pyruvate kinase was protected only by a low concentration of Mg2+; this was not the case with the M-type enzyme. Modification of the L-type form resulted in the incorporation of 1.54 +/- 0.057 mol of dye/mol of enzyme subunit in the absence of Mg2+, but only 0.73 +/- 0.024 mol of dye/mol of enzyme subunit in the presence of Mg2+. Tryptic peptide mapping of L-type pyruvate kinase modified in the presence and in the absence of Mg2+ further indicated that there were two sites modified in the enzyme, one of which was protected by Mg2+. The pKa of the nucleophile involved in the modification was calculated to be 7.1, implicating the possible involvement of a histidine residue. L-type enzyme was bound to Sepharose-immobilized Procion Blue MX-R specifically in the presence of Mg2+, whereas binding of the M-type enzyme was Mg2+-independent. The specific interaction of L-type pyruvate kinase with the dye was exploited in the large-scale purification of the enzyme and in the isolation of the phosphorylated enzyme.

Affinity Labels↗
PubMed1968

Regulation of rat liver pyruvate kinase. The effect of preincubation, pH, copper ions, fructose 1,6-diphosphate and dietary changes on enzyme activity.

1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25 degrees for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms L(A) and L(B). It is postulated that form L(A) has a low K(m) for phosphoenolpyruvate (about 0.1mm) and is not allosterically activated, whereas form L(B) is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form L(B) gives Michaelis-Menten kinetics with K(m) less than 0.1mm. It is further postulated that preincubation converts form L(A) into form L(B). 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu(2+) was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.

Animals↗
PubMed1988

Photoaffinity labeling of pyruvate kinase from rabbit muscle.

Several studies have been performed on the structure of muscle pyruvate kinase. X-ray diffraction has provided a three-dimensional picture of the active site, and chemical modification studies have revealed essential amino acid residues for substrate binding or catalysis. We have shown that 8-azido-ADP (N3 ADP) behaves as a photoaffinity label for the enzyme. This reagent upon irradiation produces inactivation of the enzyme, and the activity loss is protected by nucleotides. The partially modified enzyme shows the same Km for ADP as the native one suggesting an "all or none" inactivation effect. The incorporation of 1 mole of 14C-N3 ADP per subunit correlates with complete inactivation. A radioactive peptide was isolated from the enzyme labeled with 14C-N3 ADP. The partial sequence of this peptide showed that it corresponds to the same peptide isolated from rabbit muscle pyruvate kinase labeled with dialdehyde-ADP and with trinitrobenzenesulfonate. This peptide is identical to a region in the cat and chicken muscle enzymes, and also a high degree of homology is found in a region of the rat liver and yeast enzymes. These studies show that N3 ADP binds to the same site as dialdehyde-ADP in rabbit muscle pyruvate kinase, and this site seems to be the nucleotide binding site.

Adenosine Triphosphatases↗
PubMed1990

Diagnosis of pyruvate kinase deficiency in a transfusion-dependent patient with severe hemolytic anemia.

In a 2-yr old girl a hemolytic anemia was present since birth requiring multiple blood transfusions. Pyruvate kinase deficiency was suspected on the basis of a marginal enzyme activity, but could not be established due to the presence of massive numbers of donor cells in her peripheral blood. However, by density fractionation we succeeded in the isolation of a small fraction of the patient's own cells, in which a severe pyruvate kinase deficiency could be detected. In contrast hexokinase and glucose-6-phosphate dehydrogenase activities were extremely high, which is indicative that a very immature cell population is present in this fraction. In immunofluorescence studies a clear crossreaction was apparent with anti M2-type pyruvate kinase antibodies, whereas only a faint reaction with anti L-type could be detected. Despite the presence of a slight amount of L-type immunoreactive material, the residual activity in the patient's cell fraction could only be attributed to M2-type pyruvate kinase as was shown by cellulose acetate electrophoresis.

Anemia, Hemolytic↗
PubMed1981

Specific spectrophotometric assay for the M isoenzymes of pyruvate kinase in plasma samples containing mixtures of the muscle (M) and liver (L) isoenzymes.

Plasma pyruvate kinase (EC 2.7.1.40) activity contains two major isoenzymes. The "L" type originates from liver (erythrocytes contain a similar isoenzyme), the "M" type predominantly from skeletal muscle. The diagnostic use of pyruvate kinase as an index of muscle leakage requires an assay of only the M type. An M-type-selective assay for mixtures of the two isoenzymes was devised, based on the differences that the L form has a higher Km for phosphoenolpyruvate and is inhibited by about 95% by a high concentration of ADP. All relevant conditions of the assay have been examined and the reaction system has been optimized at 30 degrees C. The assay yields the expected Km values for M type pyruvate kinase. An apparent increase in activity in hemolysed plasma samples was found to be derived from leukocytes.

Blood Specimen Collection↗
PubMed1994

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.

Amino Acid Sequence↗
PubMed1981

Purification of a Ca2+-activated protease from rat erythrocytes and its possible effect on pyruvate kinase in vivo.

A Ca2+-activated protease with [32P]phosphopyruvate kinase as substrate was purified to about 50% from rat erythrocytes. The purification involved chromatography on Sepharose/Sephadex gels, DEAE-cellulose and (NH4)2SO4 precipitation. The protease required 3.3 mM Ca2+ for full activity. When pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was purified from erythrocytes incubated with [32P]phosphate it contained 0.5 mol [32P]phosphate/mol enzyme subunit. When 3.3 mM Ca2+ were added at hemolysis this incorporation decreased. The possible importance of this Ca2+-activated protease for the regulation of pyruvate kinase in erythrocytes is discussed.

Animals↗
PubMed1980

Hemolytic anemia and pyruvate kinase deficiency in pregnancy.

Hemolytic anemia due to deficiency of erythrocytic pyruvate kinase is a rare autosomal recessive disorder. Pregnancy complicated by pyruvate kinase deficiency is rare; there are only 8 reported cases in the literature. A case is described that was characterized by increased hemolysis during pregnancy, requiring blood transfusions before and after delivery, and complicated by severe preeclampsia at term. Fetal and maternal outcome was successful. Increased hemolysis and favorable perinatal outcome occurred in all 8 reported cases.

Adolescent↗
PubMed1992

Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells.

As a common characteristic of tumor cells, as well as of normal proliferating cells in the G1-phase of cell cycle, one finds constitutive high levels of all the glycolytic metabolites arising between glucose 6-phosphate and phosphoenolpyruvate. Thus, it is that the phosphometabolites fructose 1,6-bisphosphate, ribose 5-P, P-ribose-PP, NAD, GTP, CTO, UTP, UDP-glucose, glycerol 3-P, glycerol phosphocholine and glycerol phosphoethanolamine are useful in the 31P-nuclear magnetic resonance (NMR) detection of solid tumors in animals and man. This expansion of phosphometabolites is achieved during tumor formation as a result of reductions in levels of enzymes degrading phosphometabolites, owing to the decline in the glycerol 3-P hydrogen shuttle, and as a consequence of alterations in the glycolytic isoenzyme equipment. Tumor cells typically express a particular isoenzyme of pyruvate kinase called type M2 (K) at high levels. This isoenzyme is subject to a complex regulation by amino acids, by fructose 1,6-bisphosphate, and by hormonal- and oncogene-dependent phosphorylation. Pyruvate kinase type M2 is a substrate for the oncogene encoded PP60v-src-tyrosine kinase. A drastic decrease in the affinity for its substrate phosphoenolpyruvate found after transformation by the src-oncogene can be explained as a consequence of the phosphorylation of pyruvate kinase in serine and tyrosine. These phosphorylations induce the breakdown of tetrameric pyruvate kinase to the trimeric and dimeric forms. Unlike the tetrameric form, the dimeric form as a low affinity for phosphoenolpyruvate. Partial inactivation of pyruvate kinase and enolase on the one hand, and a hyperactivation of hexokinase and phosphofructokinase on the other hand, lead to an expansion of all metabolites. Only when these metabolites attain high levels, thereby assuring a sufficient supply of metabolites for RNA, DNA, lipid, and complex carbohydrate synthesis, can cell proliferation proceed. This accumulation of metabolites in the G1-phase cells has been termed a "metabolic budget system" because it senses not only the actual nutrient levels, but also the supply over a period of time. Monoclonal antibodies specific for the dimeric form of pyruvate kinase type M2 can be used for the immunohistological detection of tumor cells. The amount of the dimeric form in tumor cells closely correlates with the degree of malignancy and can be used for a nonspecific detection of tumors based on assays performed with patient's plasma.

Adenosine Triphosphate↗
PubMed1989

Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase.

A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has approximately 5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent Ki values of T3, L-thyroxine, and D-T3 are 30 nM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.

Amino Acid Sequence↗
PubMed1976

Salicylate-induced injury of pyruvate-kinase-deficient erythrocytes.

Salicylate is known to uncouple mitochondrial oxidative phosphorylation. Since the viability of pyruvate-kinase-deficient reticulocytes depends on ATP generated by mitochondrial metabolism, this study examined the effects of salicylate on erythrocytes deficient in pyruvate kinase. When deficient erythrocytes from patients with severe hemolysis were incubated with salicylate (2 to 30 mg per deciliter), there was a marked decrease (25 to 75 percent) in ATP. In addition, this drug-induced ATP depletion produced cell potassium and water loss, and the normal oxidant responsiveness of the hexose-monophosphate shunt was blunted. Since these cellular abnormalities are associated with accelerated hemolysis in vivo, the data suggest that aspirin therapy may aggravate hemolysis in patients with pyruvate kinase deficiency whose erythrocyte manifest sensitivity to salicylate in vitro.

Adenosine Triphosphate↗
PubMed1990

[Study of pH-dependence of kinetic parameters of pyruvate kinase from bovine adrenal cortex].

The effect of pH on the main kinetic parameters of pyruvate kinase function was studied. The maximal rate of the reaction as well as the values of Km for ADP and Ki for phenylalanine depend on pH and show a well-defined extremum at pH 6.8-7.0. Spectrofluorimetric titration of pyruvate kinase results in pH dependencies of changes in the fluorescence spectra parameters (e.g., quantum yield, half-width and position of the maximum). This enabled to determine the pH regions corresponding to changes in the state of tryptophan residues. Data from the enzyme inhibition by phenylalanine suggest that acidification of the medium leads to the decrease of the catalytic activity due to the protonation of the ionogenic group of the enzyme. Within the pH range of 7.0-8.0, the decrease of the pyruvate kinase activity is due to structural shifts in the enzyme molecule, as a result of which the steric complementariness of the enzyme active center with respect to the substrate (Mg.ADP) is impaired.

Adrenal Cortex↗
PubMed1984

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding.

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.

Alanine↗
PubMed1976

Hysteretic response of human erythrocyte pyruvate kinase to phosphoenolpyruvate. Potential role in regulation of 2,3-bisphosphoglycerate metabolism.

Human erythrocyte pyruvate kinase (EC 2.7.1.40, ATP-pyruvate phosphotransferase) was found to display a time-dependent activation (lag phase) in the reaction progress curves. The extent of this lag phase depended upon the treatment of the enzyme prior to assay. Preincubation of the enzyme with adenine nucleotides amplified the lag, whereas prior treatment with phosphoenolpyruvate diminished it. The activation process was first order in enzyme with the pseudo first order rate constants being a hyperbolic function of phosphoenolpyruvate concentration. The data provide evidence for a phosphoenol-pyruvate-mediated conversion of the enzyme to a more active form. Studies with the irreversible sulfhydryl inhibitor, N-ethylmaleimide (MalNEt), provided additional evidence for different conformational states of the enzyme induced by its substrates and effectors. Adenine nucleotides were found to promote inactivation by MalNEt and phosphoenolpyruvate protected against MalNEt. The possible metabolic significance of this "hysteretic" pyruvate kinase is discussed in relation to the known role of this enzyme in 2,3-bisphosphoglycerate metabolism (Rose, I.A. (1971) Exp. Eye Res. 11, 264-272).

Adenine Nucleotides↗
PubMed1977

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands.

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

Adipates↗