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PubMed1981

Thirteen cases of pyruvate kinase deficiency found in Japan.

Thirteen cases of pyruvate kinase (PK) deficiency, considered to be heterozygous for different PK mutants because of no consanguinities in their parents, were characterized by the International Committee for Standardization in Haematology (ICSH) recommended methods. These deficiency cases are named PK "Kagoshima," PK "Kyoto," PK "Takamatsu," PK "Abeno," PK "Kobe," PK "Marugame," PK "Hoenzaka," PK "Osaka," PK "Motomachi," PK "Gifu," PK "Hiroshima" PK "Matsumoto," and PK "Tama." The characteristics of mutant PK enzymes suggest that the cause of chronic hemolysis depends mainly on decreased affinity for phosphoenolpyruvate, thermolability, increased inhibition by adenosine triphosphate, and low activation by fructose-1, 6-diphosphate.

Adolescent↗
PubMed2003

Pyruvate kinase: current status of regulatory and functional properties.

Pyruvate kinase (PK) is a key enzyme for the glycolytic pathway and carbon metabolism in general. On the basis of the relevance and enormous diverse properties of this enzyme, this paper describes the results of a current and extensive review that determines the sites of conservation and/or difference in PK sequences, and the differences in the functional and regulatory properties of the enzymes. An alignment and analysis of 50 PK sequences from different sources and a phylogenetic tree are presented. This analysis was performed with reference to crystallographically characterized PK principally from E. coli, cat and rabbit muscle. A number of attributes of the enzyme that make it of particular interest in biomedicine and industry are also discussed.

Adenosine Diphosphate↗
PubMed1975

Erythrocyte pyruvate kinase activity in patients with haematological malignancies.

Erythrocyte pyruvate kinase (PK) activity was decreased with acute myelomonoblastic leukaemia (AML) and lymphosarcoma cell leukaemia (LSAL) but increased in patients with chronic myelocytic leukaemia (CML). There was no difference in the enzyme activity between controls and relatives of patients with AML. PK activity in patients with ALL during complete remission was higher than during untreated and relapsed stages. These data provided evidence that the altered enzyme activity is acquired rather than genetic. There was a positive correlation between PK activity and red cell creatine levels. The red cell creatine was low in patients with AML but high in patients with CML. This suggests that the altered enzyme activity in these patients is at least part due to red cell age.

Adolescent↗
PubMed1988

Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate.

The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP. In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used. The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate. Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated. In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP. The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed. The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate.

Enzyme Activation↗
PubMed1986

The effect of storage on the kinetic properties of sheep hepatic pyruvate kinase.

The kinetic properties of purified sheep hepatic pyruvate kinase change upon storage. Assayed at 0.5 mM fructose-1,6-diphosphate and 2 mM ADP, saturation of fresh enzyme with phosphoenolpyruvate is hyperbolic, with KPEP = 0.1 mM (pH 7.5, and 30 degrees C). Under similar conditions enzyme stored at -20 degrees C for 1 week or more yields a nonlinear Lineweaver-Burk plot for PEP. The data may be accounted for by the appearance of two enzymic forms with identical turnover numbers, but different KPEP (0.035 +/- 0.005 and 12.4 +/- 0.6 mM). Storage also increases the concentration of fructose-1,6-diphosphate required for maximal activation from nanomolar to millimolar levels. Assayed at 2 mM ADP and 2 mM PEP, the apparent KFDP is 10 mM. Preincubation of stored enzyme with PEP in the presence of mercaptoethanol leads to significant reversion to original kinetic properties. Available data suggest that the storage-dependent change in kinetic behavior rises from changes in subunit conformation and not from dissociation into subunits.

Adenosine Diphosphate↗
PubMed2005

Red cell pyruvate kinase deficiency: molecular and clinical aspects.

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of the glycolytic pathway causing hereditary non-spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anaemia and jaundice necessitating exchange transfusions. Erythrocyte PK is synthesized under the control of the PK-LR gene located on chromosome 1. To date, more than 150 different mutations in the PK-LR gene have been associated with PK deficiency. First attempts to delineate the biochemical and clinical consequences of the molecular defect were mainly based on the observation of the few homozygous patients and on the analysis of the three-dimensional structure of the enzyme. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1984

Maturation of the fetal lung III. Effect of transplacental TRH and 2'-thiourea treatment on phosphatidic acid phosphatase and pyruvate kinase activity in rat lung.

The activity of pyruvate kinase (PK), an enzyme playing a key role in glycolysis, was studied in the lung of Wistar R/A rats in the prenatal, postnatal and adult periods of life. The highest level was measured on the 20th gestational day, a value nearly double the adult mean. In order to elicit fetal hypothyroidism, pregnant rats were treated with 0.1% thiourea solution from the 14th day of gestation up to delivery. The animals were killed on the 21st or 22nd gestational day or on the 2nd postnatal day. A significant increase in PK activity was seen in the treated animals as compared to the control group, while the activity of phosphatidic acid phosphatase (PAPase) remained unchanged. Fetal hyperthyroidism was induced by intravenous injections of TRH to the pregnant mother rat. This resulted in an increase of PAPase and a decrease of PK activity as compared to the control group. The difference was statistically significant in all instances, the level of significance, however, depended on the time when treatment had been initiated.

Administration, Oral↗
PubMed2002

Molecular and regulatory properties of leucoplast pyruvate kinase from Brassica napus (rapeseed) suspension cells.

Plastidic pyruvate kinase (PK(p)) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 micromol phosphoenolpyruvate (PEP) utilized/min/mg protein. SDS-PAGE, immunoblot and gel filtration analyses indicated that this PK(p) exists as a 380-kDa heterohexamer composed of equal proportions of 64- (alpha-subunit) and 58-kDa (beta-subunit) polypeptides. The N-terminal sequence of the PK(p) alpha- and beta-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively. B. napus PK(p) displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (K(m) = 0.052 and 0.14 mM, respectively). 6-Phosphogluconate functioned as an activator (K(a) = 0.12 mM) by increasing V(max) by approximately 35% while decreasing the K(m)(PEP) and K(m)(ADP) values by 40 and 50%, respectively. 2-Oxoglutarate and oxalate were the most effective inhibitors (I(50) = 8.3 and 0.23 mM, respectively). A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B. napus leucoplasts.

Adenosine Triphosphate↗
PubMed1991

Isolation and characterization of the human pyruvate kinase M gene.

Genomic clones containing the human pyruvate kinase M (PKM) gene, which encodes the M1-type and M2-type isozymes, were isolated and their exon sequences were determined. The gene is approximately 32 kb and consists of 12 exons and 11 introns. Exons 9 and 10 contain sequences specific to the M1 and M2 types, respectively, indicating that the human isozymes are produced from the same gene by alternative splicing as in the case of the rat gene. The exon-intron structure of the human PKM gene is identical to that of the rat gene, and the introns of both genes interrupt the exons at the same points. Introns 6 and 7 begin with GC dinucleotide instead of the consensus GT, but the other exon-intron boundaries are consistent with the GT-AG rule. The gene is transcribed from multiple start sites. The 5'-flanking region of the gene contains putative Sp1-binding sites, but no TATA box or CAAT box, and shows high sequence similarity to that of the rat M gene. Bacterial chloramphenicol acetyltransferase assay revealed that the upstream region between positions -493 and -51 contained a cis-acting element(s) that was essential for expression of the M gene in HeLa cells. Long stretches of conserved regions were found in the introns around the M1-specific and M2-specific exons, suggesting that these regions may be involved in the alternative splicing machinery.

Amino Acid Sequence↗
PubMed1993

Functional characterization of the L-type pyruvate kinase gene glucose response complex.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Animals↗
PubMed2003

[Usefulness of plasma tumor M2-pyruvate kinase in the diagnosis of gastrointestinal cancer].

BACKGROUND/AIMS: Pyruvate kinase (PK) is a key enzyme of glycolysis. Different isoforms of this enzyme are tissue-specifically expressed (M2-PK, M1-PK, R-PK, L-PK). The concentration of the dimeric M2-PK is increased in a metabolic state of tumor cells. In this case, the dimeric M2-PK is termed Tumor M2-PK. We investigated EDTA-plasma of 73 patients with gastrointestinal (GI) cancer and 61 healthy controls to evaluate its significance in diagnosing GI cancer. METHODS: Plasma Tumor M2-PK was measured using an ELISA assay based on two monoclonal antibodies which specifically react with the dimeric Tumor M2-PK. RESULTS: The sensitivity of Tumor M2-PK was 67.1% for all GI cancers, that of CA 19-9 was 38.4% and that of CEA was 34.3%. The specificity of Tumor M2-PK was 91.8% (cutoff=20 U/mL). Tumor M2-PK showed a high sensitivity in gastric cancer (62.2%), colorectal cancer (66.7%) and bile duct cancer (75.0%). In colorectal cancer, the combination of Tumor M2-PK with CEA resulted in a remarkable increase in the sensitivity (86.2%). The average Tumor M2-PK levels were generally elevated in the metastatic GI cancer patients compared to nonmetastatic patients, especially in stomach cancer with statistical significance (p=0.005). CONCLUSIONS: Tumor M2-PK in EDTA-plasma seems to be a new valuable tumor marker in GI cancer.

Adult↗
PubMed1992

In vitro and in vivo protein--DNA interactions on the rat erythroid-specific L' pyruvate kinase gene promoter.

The rat L-type pyruvate kinase gene possesses two alternative tissue-specific promoters, located 472 bp apart; the upstream L' promoter is erythroid-specific and the downstream L promoter is hepatocyte-specific. The erythroid-specific L' promoter is strongly active in fetal liver at day 17 of gestation, while its activity rapidly decreases thereafter. A L' promoter fragment spanning from nucleotide -320 to +10 with respect to the cap site is able to direct a weak but erythroid-specific transcription in a cell-free system. We have used DNAse I footprinting and gel mobility shift assays to characterize and identify the binding of nuclear factors from both 17-day-old fetal liver and adult liver nuclear extracts to a 320 bp fragment of the 5' flanking region of the gene in vitro. Two clusters of erythroid-specific interactions were found. The proximal cluster consists of two GATA-1 binding sites at -50 bp and -65 bp from the transcription initiation site, immediately downstream of a CACC motif and two G/C-rich elements. The distal cluster of cis-elements, located 130 bp upstream, corresponds to two GATA-1 sequences. These two sequences overlap NF1 motifs interacting with ubiquitous NF1 transcriptional factors in presence of adult hepatic extracts. Furthermore, we have examined in vivo protein-DNA interactions by DMS footprinting in livers of 17-day-old rat fetuses and adult rats. We found that the sites characterized in vitro are occupied in vivo. Therefore, in adult liver the L' promoter, although inactive, nevertheless interacts with ubiquitous factors.

Aging↗
PubMed1998

The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Allosteric Regulation↗
PubMed2000

Evidence against the peroxisome proliferator-activated receptor alpha (PPARalpha) as the mediator for polyunsaturated fatty acid suppression of hepatic L-pyruvate kinase gene transcription.

The glycolytic enzyme, L-pyruvate kinase (L-PK), plays an important role in hepatic glucose metabolism. Insulin and glucose induce L-PK gene expression, while glucagon and polyunsaturated fatty acids (PUFA) inhibit L-PK gene expression. We have been interested in defining the PUFA regulation of L-PK. The cis-regulatory target for PUFA action includes an imperfect direct repeat (DR1) that binds HNF-4. HNF4 plays an ancillary role in the insulin/glucose-mediated transactivation of the L-PK gene. Because the fatty acid-activated nuclear receptor, peroxisome proliferator-activated receptor (PPARalpha), binds DR1-like elements and has been reported to interfere with HNF4 action, we examined the role PPARalpha plays in the regulation of L-PK gene transcription. Feeding rats either fish oil or the potent PPARalpha activator, WY14,643, suppressed rat hepatic L-PK mRNA and gene transcription. The PPARalpha-null mouse was used to evaluate the role of the PPARalpha in hepatic transcriptional control of L-PK. While WY14,643 control of L-PK gene expression required the PPARalpha, PUFA regulation of L-PK gene expression was independent of the PPARalpha. Transfection studies in cultured primary hepatocytes localized the cis-regulatory target for WY14,643/PPARalpha action to the L-PK HNF4 binding site. However, PPARalpha/RXRalpha heterodimers did not bind this region. Although both WY14,643 and PUFA suppress L-PK gene transcription through the same element, PUFA regulation of L-PK does not require the PPARalpha and PPARalpha/RXRalpha does not bind the L-PK promoter. These studies suggest that other intermediary factors are involved in both the PUFA and PPARalpha regulation of L-PK gene transcription.

Animals↗
PubMed1976

Kinetic properties of pyruvate kinase in human maternal leukocytes in fetal malnutrition.

Pyruvate kinase (PK) is one of the regulatory enzymes in glycolysis. The present study was undertaken to determine whether regulation of the enzyme by normally occurring metabolites was disturbed in leukocytes of mothers who delivered fetally malnourished (FM) babies. Kinetic studies of enzyme regulation by physiologic effectors approximated a potential regulating mechanism of the enzyme in its cellular environment. There are two isoenzymes of PK. Leukocytes contain an M2 enzyme with intermediate regulatory properties between the liver (type L) and the muscle (type M) enzymes. The presence in the cell of M2 PK in the A form leads to inhibition of glycolysis by amino acids, such as alanine, and therefore, to the sparing of glucose but probably inhibition of energy production from glucose. In this study, leukocytes were isolated from blood of six pregnant women and 11 women in the postpartum period in Oklahoma and at parturition from 31 women in Mexico. Fourteen of the latter group delivered FM babies. The kinetic characteristics of the nonpurified enzyme PK with respect to allosteric modulation in fructose-1, 6-phosphate (FDP) and L-alanine (Ala) were studied in the leukocyte extracts. Data for initial reaction velocities (v) vs substrate concentrations (s), double reciprocal Lineweaver-Burk plots, and Hill plots are presented. The equations for the double reciprocal plots were determined by linear regression analysis. The enzyme constants were derived by computer, and the values compared by the Mann-Whitney U-test. In all subjects studied, 0.5 mM FDP activated and 2 mM L-alanine inhibited the enzyme. During pregnancy, the v vs s concentration curves were hyperbolic (Hill coefficient, n is less than 1.0) except for the Ala-inhibited enzyme during pregnancy, which had a sigmoid curve, n=1.54. The interaction of FDP and Ala was dependent on the concentration of the substrate phosphoenolpyruvate (PEP) at low [PEP]. There was net activation, not inhibition, at high concentrations; the switchover was at 0.5 mM PEP during pregnancy. In Mexican mothers having normal babies (normal mothers) the maximum initial velocity, V (micromoles per min per mg of DNA), with respect to PEP, was 2.22+/-0.34; in FM mothers, V was 2.01+/-0.44. With respect to binding of the substrate, PEP, V of the leukocyte enzymes in FM mothers vs normal mothers was equally inhibited by Ala (deltaV=-50% vs -47%), but was significantly less responsive to stimulation by FDP (deltaV=+10% vs +75%). When both Ala and FDP were present, FDP less effectively overcame the inhibition by Ala (deltaV=-9% vs +54%). The K0.05 of the enzyme (molar concentration X 10(-4) PEP) was significantly reduced by FDP, whether Ala was present or not, during pregnancy and in the postpartum period in leukocytes of Oklahoma mothers and at term in Mexican mothers. The K0.5 for normal and FM mothers was similar. Thus, the enzyme in leukocytes of Mexican mothers who delivered FM and normal babies exhibited different kinetic responses to the allosteric modulators...

Alanine↗
PubMed1998

Purification and characterization of pyruvate kinase from Schizosaccharomyces pombe: evidence for an unusual quaternary structure.

Earlier attempts to purify and characterize nonrecombinant pyruvate kinase from Schizosaccharomyces pombe proved difficult due to problems associated with the instability of the protein. The enzyme has been overexpressed in Saccharomyces cerevisiae strain AH22, permitting studies to determine the conditions required to stabilize the enzyme during purification. Recombinant S. pombe pyruvate kinase was purified by a combination of ion-exchange chromatography and gel filtration. The purified enzyme showed sigmoidal kinetics with respect to PEP; in the presence of FBP, the kinetics were restored to Michaelis-Menten behavior. With respect to ADP, the Hill coefficient was not affected by FBP. Determination of the molecular mass of the purified enzyme by ultracentrifugation showed that it behaved as a dimer-tetramer system with a Kd of approximately 1 microM.

Adenosine Diphosphate↗
PubMed1982

Regulation of rat liver L-type pyruvate kinase mRNA by insulin and by fructose.

The effects have been studied of streptozotocin-induced diabetes and subsequent insulin administration or feeding of a high fructose diet on the amount of enzyme protein and mRNA activity of L-type pyruvate kinase in rat liver. Diabetes markedly decreased the L-type enzyme activity in rat liver and insulin treatment resulted in restoration of the enzyme activity to normal. A high fructose diet also increased the enzyme activity in diabetic rats but to a lesser extent. Immunochemical analysis showed that these alterations in the enzyme activity were due to changes in the amount of immunoreactive enzyme protein. The mechanism of the changes was studied further by assaying the level of functional mRNA coding for this enzyme in a nuclease-treated reticulocyte lysate system with total RNA isolated from rat liver. L-type pyruvate kinase mRNA, expressed as a percentage of the total protein synthesized, was greatly decreased in diabetic rats. Insulin administration resulted in recovery of the mRNA activity to the normal level within 24 h. The lag period before accumulation of translatable mRNA of the L-type enzyme was about 4-5 h. The mRNA activity was also increased in diabetic rats fed a high fructose diet. This fructose effect, which was much smaller than the insulin effect, was maximal after feeding fructose diet for one day. These changes were approximately comparable to the changes in enzyme activity. Thus, it is suggested that regulations of rat liver L-type pyruvate kinase by insulin and by fructose are primarily due to changes in the level of translatable mRNA of this enzyme.

Animals↗
PubMedDate not supplied

13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis.

When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.

Bacillus subtilis↗