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PubMed1999

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients.

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G --> C on exon 12 and two 5' splice donor site (GT) mutations on intron 8 [IVS8(+2)T --> G] and intron 10 [IVS10(+1)G --> C]. Two previously described missense mutations, 1456C --> T and 993C --> A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G --> C or IVS8(+2)T --> G.

Adult↗

PubMed1984

N-hydroxycarbamate is the substrate for the pyruvate kinase catalyzed phosphorylation of hydroxylamine.

The true substrate for the pyruvate kinase catalyzed phosphorylation of hydroxylamine at high pH which is activated by bicarbonate is shown to be N-hydroxycarbamate, since a lag is seen when the reaction is started by the addition of bicarbonate or hydroxylamine but a burst appears when it is started with a mixture of the two. The lag can be diminished by addition of carbonic anhydrase but not eliminated, showing that CO2 is an intermediate in the formation of the carbamate and that both the formation of CO2 and the subsequent reaction of CO2 with hydroxylamine limit the rate of carbamate formation. The equilibrium constant for the reaction bicarbonate + hydroxylamine reversed N-hydroxycarbamate is 1.33 M-1. The product of the phosphorylation decomposes by loss of CO2 to O-phosphorylhydroxylamine, which is stable at 25 degrees C between pH 3 and 11 and has pK2 = 5.63 for the phosphate and pK3 = 10.26 for the amino group.

Bicarbonates↗

PubMed1993

Rapid determination of erythrocyte pyruvate kinase activity.

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.

Adolescent↗

PubMed1988

Impaired erythrocyte phosphoribosylpyrophosphate formation in hemolytic anemia due to pyruvate kinase deficiency.

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.

2,3-Diphosphoglycerate↗

PubMed2006

Mining for allosteric information: natural mutations and positional sequence conservation in pyruvate kinase.

Although the amino acid sequences and the structures of pyruvate kinase (PYK) isozymes are highly conserved, allosteric regulations differ. This suggests that amino acids with low conservation play important roles in the allosteric mechanism. The current work exploits a 'natural screen'- the 122 point mutations identified in the human gene encoding the erythrocyte PYK isozyme and associated with nonspherocytic hemolytic anemia - to learn what amino acid positions in PYK may be important for allosteric regulations. In addition to the mutations, we consider the conservation of each amino acid position across 241 PYK sequences. Three groups of residue positions have been created, those with: (1) no disease causing mutation identified; (2) a disease causing mutation identified and high conservation across isozymes; and (3) a disease causing mutation identified and low conservation. Mutations at positions not identified in the natural screen are likely to be tolerated with minimal loss of function. Mutations at highly conserved positions are more likely to disrupt properties common to all PYK isozymes (e.g., structure, catalysis). Residues in the third group are likely to be involved in roles that are necessary for function but not common to all isozymes (e.g., allostery). Many of the Group 3 residues are located in the C-domain and to a lesser extent the A domain.

Allosteric Regulation↗

PubMed1995

Functional synergism in the carbohydrate-induced activation of liver-type pyruvate kinase gene expression.

Hepatic expression of the liver-type pyruvate kinase (L-PK) gene is induced at the transcriptional level by increased carbohydrate metabolism in the rat. The carbohydrate response of the L-PK gene requires sequences from -171 to -124, which encompass adjacent major late transcription factor (MLTF)-like and hepatic nuclear factor (HNF)-4 binding sites. Neither site alone is capable of conferring a response, prompting us to explore the mechanism of synergy between the MLTF-like factor and HNF-4. Spacing requirements between the two factor binding sites were tested by generating a series of mutations that altered the distance between these sites. Surprisingly, all of the constructs with spacing mutations were capable of responding to elevated glucose when introduced into primary hepatocytes. Thus the glucose response does not depend on the rigid phasing of the MLTF-like and HNF-4 factors, suggesting that the factors binding to these two sites do not interact directly with each other. Substitution or inversion of the PK HNF-4 site abrogated the response to glucose and also significantly suppressed the promoter activity under non-inducing conditions. We conclude that the MLTF-like factor and HNF-4 co-operate functionally to maintain the basal activity, as well as the carbohydrate responsiveness, of the L-PK gene. A mechanism other than co-operative DNA binding is responsible for the synergism.

Animals↗

PubMed1986

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Animals↗

PubMed1995

Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.

BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation. RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme. CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.

Allosteric Regulation↗

PubMed1998

Structure of the bis(Mg2+)-ATP-oxalate complex of the rabbit muscle pyruvate kinase at 2.1 A resolution: ATP binding over a barrel.

Pyruvate kinase from rabbit muscle has been cocrystallized as a complex with MgIIATP, oxalate, Mg2+, and either K+ or Na+. Crystals with either Na+ or K+ belong to the space group P2(1)2(1)2(1), and the asymmetric units contain two tetramers. The structures were solved by molecular replacement and refined to 2.1 (K+) and 2.35 A (Na+) resolution. The structures of the Na+ and K+ complexes are virtually isomorphous. Each of the eight subunits within the asymmetric unit contains MgIIoxalate as a bidentate complex linked to the protein through coordination of Mg2+ to the carboxylates of Glu 271 and Asp 295. Six of the subunits also contain an alpha,beta,gamma-tridentate complex of MgIIATP, and the active-site cleft, located between domains A and B, is closed in these subunits. In the remaining two subunits MgIIATP is missing, and the active-site cleft is open. Closure of the active-site cleft in the fully liganded subunits includes a rotation of 41 degrees of the B domain relative to the A domain. alpha-Carbons of residues in the B domain undergo movements of up to 17.8 A (Lys 124) in the cleft closure. Lys 206, Arg 119, and Asp 177 from the B domain move several angstroms from their positions in the open conformation to contact the MgIIATP complex in the active site. The gamma-phosphate of ATP coordinates to both magnesium ions and to the monovalent cation, K+ or Na+. A Mg2+-coordinated oxygen from the MgIIoxalate complex lies 3.0 A from Pgamma of ATP, and this oxygen is positioned for an in-line attack on the phosphorus. The side chains of Lys 269 and Arg 119 are positioned to provide leaving-group activation in the forward and reverse directions. There is no obvious candidate for the acid/base catalyst near the 2-si face of the prospective enolate of the normal substrate. A functional group linked through solvent and side-chain hydroxyls may function in a proton relay.

Adenosine Triphosphate↗

PubMed1991

Alteration in L-type pyruvate kinase gene expression is not associated with the LF-B1 mRNA level.

The relation of expression of the LF-B1 gene with the L-type pyruvate kinase (L-PK) mRNA level in rat liver and hepatoma cells was investigated. The L-PK mRNA level in rat liver changed after partial hepatectomy, during development and on intake of a high carbohydrate diet, while the level of LF-B1 mRNA remained unchanged or altered reciprocally. Dedifferentiated AH-130 cells, which did not express L-PK mRNA, expressed LF-B1 mRNA. These results suggest that transcription of the pyruvate kinase L gene is not simply regulated by the level of LF-B1 mRNA.

Animals↗

PubMed1983

Pyruvate kinase type M2 is phosphorylated in the intact chicken liver cell.

Isolated hepatocytes from 24 h starved chicks were depleted of phosphate and incubated in the presence of 32P-orthophosphate. Pyruvate kinase type M2 from crude cell extracts was partially purified by chromatography on DEAE-Sephacel and hydroxylapatite. SDS slab gel electrophoresis of the fractions containing the enzyme and immunoprecipitation with antisera showed the phosphorylation of pyruvate kinase type M2. Phosphoamino acid analysis identified serine as phosphate acceptor.

Amino Acids↗

PubMedDate not supplied

Pyruvate kinase deficiency: an unusual cause of puerperal jaundice.

We present one of the rarer causes of pregnancy-associated jaundice in the immediate puerperium. A 30-year-old multipara with pyruvate kinase deficiency became jaundiced within a few hours of delivery, following an uneventful pregnancy and labour at term. She responded to conservative management, the jaundice clearing and the altered liver function showing a steady return to normal. Pyruvate kinase deficiency may cause pregnancy-associated jaundice. A thorough investigation is still necessary to exclude other causes of jaundice in pregnancy and the puerperium.

Adult↗

PubMed1991

Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture.

In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.

Animals↗

PubMedDate not supplied

Molecular cloning of the gene that codes for the pyruvate kinase of Bacillus subtilis: primary characterization of a strain carrying this gene insertionally inactivated.

We have cloned and characterized the pykA gene from Bacillus subtilis which codes for a pyruvate kinase (PK) enzyme. This gene has been located downstream a putative phosphofructokinase gene, suggesting that they are part of the same operon. The deduced amino acid sequence of this PK showed a strong similarity to other PKs from different sources; however, as it has been found in other bacilli, the B. subtilis pykA enzyme had an extra C-terminal sequence consisting of about 112 amino acid residues. This gene was insertionally inactivated at the chromosomal level, with an antibiotic resistance marker. The analysis of this mutation in wild type and pts- backgrounds, indicated that B. subtilis has no other pyruvate kinase activity capable of complementing the absence of PykA.

Amino Acid Sequence↗

PubMed1985

Effect of polyethylene glycol on the kinetic behaviour of pyruvate kinase and other potentially regulatory liver enzymes.

Assay in the presence of 10% polyethylene glycol has been systematically used with potentially regulatory liver enzymes as an indirect way to induce aggregation of enzymes corresponding to that which could occur at their physiological concentrations. Pyruvate kinase L was markedly affected by polyethylene glycol, as was muscle phosphorylase a, while pyruvate kinase M as well as glucokinase, fructose-1,6-bisphosphatase and other liver enzymes examined were not affected.

Adenosine Triphosphate↗

PubMed1998

Interaction of M1 and M2 isozymes pyruvate kinase from human tissues with phospholipids.

The effect of pH and the presence of FBP on the interaction of skeletal muscle (PK-M1) and kidney or tumor meningioma (PK-M2) pyruvate kinase with the phospholipids liposomes were investigated by ultracentrifugation and steady-state kinetics and were compared with those results obtained using the bovine heart (PK-M1) isoenzyme which we previously studied. Pyruvate kinase specific activity increases upon the interaction with liposomes. The activation is specifically sensitive to presence of phosphatidylserine (PS) in liposomes. Liposomes made of phosphatidylcholine + phosphatidylserine mixture are good adsorptive systems for both human and bovine of M-type isozymes at low ionic strength. Interaction of PK-M1 with PS liposomes results in the change of Vmax and K(m) values for PEP without marked effect on Hill coefficients. Addition of PS liposomes to PK-M2 induces hyperbolic saturation curves for PEP.

Adsorption↗

PubMed1985

Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme.

Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.

Adenosine Diphosphate↗

PubMed1990

Patient with pyruvate kinase deficiency developed acute myelogenous leukemia.

A 44-year-old previously healthy male was diagnosed as anemic and treated at a nearby hospital. A year later, he was admitted to our hospital for precise examination of the progression of refractory anemia. Blood examination showed hemolytic anemia and more detailed examination of this hemolysis revealed pyruvate kinase deficiency. The activity was 15.1% of a normal control. Pyruvate kinase activities of his family members were normal or slightly decreased. Ten months after admission to our hospital, 2% of blast cells with Auer's body in the peripheral blood were noted which increased progressively. The bone marrow contained 17.3% of blast cells with a subsequent diagnosis of acute myelogenous leukemia being made. Although intensive chemotherapy was performed, the patient achieved a brief remission and died 2 years after admission to our hospital.

Adult↗