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PubMed1984

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione.

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.

Adenosine Diphosphate↗
PubMed1992

Diet-dependent carcinogenesis of pancreatic islets and liver in transgenic mice expressing oncogenes under the control of the L-type pyruvate kinase gene promoter.

The liver-type pyruvate kinase (L-PK) gene is controlled positively by insulin and carbohydrates, negatively by glucagon and fasting. Diet-inducible models of carcinogenesis were obtained using the L-PK gene promoter and regulatory sequences to control the expression of c-myc and SV40 T oncogenes in transgenic mice. L-PK/c-myc and L-PK/Tag animals fed a carbohydrate-rich diet developed hepatocarcinomas. In addition, L-PK/Tag animals developed diet-dependent, aggressive endocrine pancreatic tumors, preceded by islet hyperplasia involving the different analysed cell populations (alpha, beta and delta). Expression of the L-PK gene was demonstrated in pancreatic tumors, in rat isolated islets and in rat insulinoma-derived cells (RIN line), revealing a new tissue specificity of the L-PK gene. Our results suggest that this gene may be expressed in islet progenitor cells from which the different mature endocrine cells derive.

Animals↗
PubMed1998

Cloning and expression of the Zymomonas mobilis pyruvate kinase gene in Escherichia coli.

The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.

5' Untranslated Regions↗
PubMed1986

Tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase mRNAs.

A single L-type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 X 10(3) bases (kb). All three species are polyadenylated and translatable into the same L-type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5' ends. Nuclease S1 protection experiments with various cDNA and 3' genomic probes indicate that the different mRNA species only differ by the length of their 3' noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run-on assays with specific probes recognizing only the 3.2-kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3' from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue-specific and changes with development. The role of an 'identifier sequence' located in the 3' noncoding sequence of the 3.2-kb species in such a tissue-specific use of alternative polyadenylation sites is discussed.

Animals↗
PubMed1994

Respective roles of glucose, fructose, and insulin in the regulation of the liver-specific pyruvate kinase gene promoter.

The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.

Animals↗
PubMed1994

The molecular basis of canine pyruvate kinase deficiency.

Inherited hemolytic anemia due to pyruvate kinase (PK) deficiency is an autosomal recessive disease of the Basenji dog that closely resembles human PK deficiency. Characterization of transcriptional and translational expression of PK isozymes and sequencing of DNA from normal and mutant dogs were performed to identify the genetic defect in Basenji dogs. Measurement of erythrocytic PK activity by ion exchange chromatography, substrate kinetics, immunologic reactivity, and electrophoretic mobility suggests that M2-type PK is the major form of PK activity in erythrocytes of PK-deficient dogs, in contrast to normal dogs having only R-type PK activity. Both R-type and M2-type PK mRNA are detectable in reticulocytes of PK-deficient dogs, suggesting that the aberrant isozyme expression is not due to a failure in the erythroid maturational switch from M2- to R-type isozymes. Nucleotide sequence data from wild-type and mutant R-type PK cDNA identified a single nucleotide deletion, delta C433, in the mutant cDNA. The deduced amino acid sequence predicts a truncated mutant protein devoid of all residues contributing to the catalytic site of the wild-type protein. In the absence of R-type PK activity, there is anomalous compensatory expression of M2-type PK in erythroid cells of PK-deficient Basenjis. The PK-deficient Basenji dog may be valuable in somatic cell gene therapy trials involving manipulation of hematopoietic stem cells.

Amino Acid Sequence↗
PubMed1989

Primary structure of three peptides at the catalytic and allosteric sites of the fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli.

Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphate. The labelled peptides have been sequenced. The comparison of their sequences with the primary structure of the cat muscle pyruvate kinase allowed to state that peptide I fits the region spanning residues 423-438 (53% identity), peptide II corresponds to residues 442-457 (44% identity) and peptide III encompasses residues 342-368 (70% identity). These findings are discussed in connection with our previous results on the involvement of the three peptides in the catalytic and regulatory properties of the enzyme (Valentini, G., Speranza, M.L., Iadarola, P., Ferri, G. & Malcovati, M. (1988) Biol. Chem. Hoppe-Seyler 369, 1219-1226) and in connection with their location in the three-dimensional structure of the cat muscle pyruvate kinase (Muirhead, H., Clayden, D.A., Lorimer, C.G., Fothergill-Gilmore, L.A., Schiltz, E. & Schmitt, W. (1986) EMBO J. 5, 475-481).

Amino Acid Sequence↗
PubMed1990

[Regulation of pyruvate kinase gene expression and its clinical application].

Pyruvate kinase (PK), an important glycolytic enzyme, has two genes per haploid genome in mammals and each gene encodes two isozymes. The L gene produces the L- and R-types using alternative promoters. The M gene generates the M1- and M2-types by alternative RNA splicing. Expression of the PK isozymes is tissue-specific and regulated developmentally. Carcinogenesis apparently reverses the developmental process. Expression of the L-type is regulated by dietary and hormonal factors. These regulations occurred at post-transcriptional as well as transcriptional levels. The transcription of hepatic L-type PK is stimulated by insulin and inhibited by glucagon. The insulin action requires ongoing protein synthesis and metabolism of glucose, and is enhanced by glucocorticoid. Dietary fructose also stimulates expression of the L-type in liver, kidney, and small intestine, but its mechanism is dependent on tissues, and on plasma insulin levels in the case of the liver. In normal liver, the fructose induction is explained by stimulation of gene transcription. On the other hand, fructose acts mainly at the post-transcriptional level in diabetic liver and other tissues. These fructose effects are attributable to common metabolite(s) of fructose and glycerol. Studies on transgenic mice indicate that the 5'-flanking region up to -3 kb of the L-type PK gene contains cis-acting elements responsible for insulin regulation and tissue-specific expression of the L-type. Further analysis using a transient expression assay revealed the presence of multiple elements necessary for expression of the L-type in hepatocytes in the region up to -170b.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.

Adenosine Diphosphate↗
PubMed1975

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.

Adenosine Monophosphate↗
PubMed1978

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates.

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

Alanine↗
PubMed1988

Heterogeneity of glycolytic enzyme activity and isozyme composition of pyruvate kinase in breast cancer.

In 6 patients with breast cancer - of whom specimens of the primary tumor as well as one of its metastases were available for examination - we demonstrated intratumoral and intertumoral heterogeneity in expression of activity of the glycolytic enzymes hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase. Heterogeneity also existed in isozyme composition of pyruvate kinase. The transition of the tumors towards normal surrounding breast tissue showed either a sharp drop in activity, or a gradual decrease in activity, corresponding to pushing margins or infiltrative growth of the tumor as was demonstrated by histologic examination of these specimens. Likewise, the shift towards expression of K isozyme of pyruvate kinase in breast cancer compared to normal breast tissue could be demonstrated.

Adult↗
PubMed1975

Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli.

Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions. Their kinetic behavior has been characteriized with respect to different effectors and substrates. The conclusions reached on one hand by Malcovati and Kornberg (Biochim. Biophys. Acta (1969) 178, 420-423), on the other hand by Fraenkel, Kotlarz and Buc (J. Biol. Chem. (1973) 248, 4865-4866) have been found to be true in aerobiosis as well as in anaerobiosis. The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions. Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time.

Aerobiosis↗
PubMed1998

Pyruvate kinase of Trypanosoma brucei: overexpression, purification, and functional characterization of wild-type and mutated enzyme.

A procedure was developed for overexpression of Trypanosoma brucei pyruvate kinase in Escherichia coli. The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higher S(0.5) for phosphoenolpyruvate and a significantly smaller kcat than the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.

Animals↗
PubMed1982

Peptide structures of pyruvate kinase isozymes: 2. Origins of types M1 and M2 isozymes suggested from species-variations in their peptide maps.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) isozymes were purified: type M1 and M2 isozymes from rat, mouse and rabbit, type M1 from bull frog and type L from rat and mouse. The amino acid composition of type M1 and M2 isozymes from various species were very similar and those of type L isozymes from rat and mouse also very similar. The NH2-terminals to type M1 and M2 from rat and mouse were (Pro-Lys-Pro-), but those of the other enzymes appeared to be blocked. The COOH-terminals of type M1 and M2 from rat, mouse and rabbit were (-Val-Pro). Comparison of tryptic peptide maps of type M1 and M2 from rat, mouse and rabbit indicated that type M1 and M2 could not be produced by post-synthesis modification, but could be encoded by different type-specific mRNAs. Comparative studies on type M1 and M2 of different species showed that type M1-specific peptide spots were highly variable, whereas type M2-specific peptide spots were highly conserved. The type L isozyme showed marked species variation, indicating that this differentiated isozyme evolved more rapidly than type M2, which may be a prototype or undifferentiated isozyme.

Amino Acid Sequence↗
PubMed1985

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate.

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.

Adenosine Diphosphate↗
PubMed1987

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.

Amino Acids↗