SEARCH · Search PubMed

Results for “Pyruvate Kinase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

PubMed1988

Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor.

Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143-420) is a regular 8-fold beta/alpha-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.

Biological Evolution↗

PubMed1977

Pyruvate kinase isozymes in cells isolated from fetal and regenerating rat liver.

There are at least three major mammalian isozymes of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40), designated K4, L4, and M4. Whereas parenchymal cells from adult rat liver contain only the type L isozyme, parenchymal cells isolated from fetal and regenerating liver were found to synthesize both the K4 and L4 isozymes. A small amount of K-M hybrid was seen in regenerating liver, but there were no detectable M-L or K-L hybrids. Thus, it appears that type L pyruvate kinase is not synthesized at the same time in the same liver cell with either of the other two isozymes. The intermediate electrophoretic bands seen with homogenates of whole fetal liver, and in some earlier work attributed to either hybrid isozymes or to the presence of M4, are contributed by nonparenchymal cells which, in the fetus, are largely hemopoietic. These additional bands of pyruvate kinase are electrophoretically and immunologically similar to the pyruvate kinase isozymes found in adult erythrocytes. The results reported here suggest a very rigorous control in the synthesis of K4 and L4 isozymes in parenchymal cells of both fetal and regenerating liver as opposed to developing neurons and glia, where the shift from synthesis of type K to type M subunits appears to occur gradually and results in the production of substantial amounts of hybrid isozymes.

Animals↗

PubMed1976

A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase.

A revised preparation of pyruvate kinase from saccharomyces cerevisiae is reported. By purifying this cold-labile enzyme at room temperature, an improved recovery and specific activity was obtained. More than 350 mg of pure enzyme with a specific activity of 350 to 400 units/mg at 30 degrees were obtained from a pound of fresh yeast. The last step of the preparation, passage of the enzyme over Sephadex G-100, was required to remove a contaminating protease. The molecular parameters of the new preparation are: molecular weight, 209,000; four subunits of identical size; E 280 nm, 0.51; pI 6.6; and pH optimum, 6.28. Kinetic parameters are: Km for P-enolpyruvate and ADP, 0.09 and 0.18 mM in the presence of saturating Fru-1,6-P2, and 1.8 and 0.34 mM in the absence of Fru-1,6-P2; Ka for Fru-1,6-P2, 0.014 mM. No free NH2-terminal amino acid could be detected. Amino acid composition was determined and compared with other pyruvate kinase preparations.

Amino Acids↗

PubMed1978

Properties of pyruvate kinase from soybean nodule cytosol.

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N(2) fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K(+) and NH(4) (+) concentrations. In the presence of physiological concentrations of K(+) and high phosphoenolpyruvate (PEP) concentrations, NH(4) (+) inhibited enzyme activity. Comparisons of kinetic parameters (V(max) and apparent K(a)) for NH(4) (+) and K(+) with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K(a) (monovalent cation) and K(m) (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH(4) (+).

Journal Article↗

PubMed1988

Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei.

The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 mumol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

Adenosine Diphosphate↗

PubMed1996

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

Adenosine Diphosphate↗

PubMed1976

Evidence of a novel role for monovalent cations in pyruvate kinase catalysis.

In the pyruvate kinase (EC 2.7.1.40) reaction, the complete enzyme-products complex consists of enzyme, pyruvate, ATP, a divalent cation, and a monovalent cation, usually K+. The dissociation of this complex can be slow permitting reversible enolization of pyruvate in this complex during the course of the forward reaction. High concentrations of each component in the enzyme-products complex inhibits the forward reaction, but only elevated concentrations of the monovalent cation decrease the net rate of product dissociation relative to that of pyruvate enolization. This result indicates that the monovalent cation can be the first component released from the enzyme-products complex and that the presence of an inhibiting monovalent cation concentration leads to re-formation of the complete complex, which is necessary for pyruvate enolization. The evidence suggests that the monovalent cation can bind and release with each turnover of the enzyme. While the data do not permit the conclusion that first release of monovalent cation is the exclusive pathway for dissociation of the enzyme-products complex, no other component once released can reassociate rapidly enough to form a complete complex during the forward reaction. Inhibition by these components must be attributed to the formation of abortive complexes.

Adenosine Triphosphate↗

PubMed1977

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.

Amino Acids↗

PubMed2002

Phosphoenolpyruvate-dependent tubulin-pyruvate kinase interaction at different organizational levels.

Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregates and bundles the tubules in a salt concentration-dependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo.

Animals↗

PubMed1999

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.

Animals↗

PubMed1995

[Diagnosis of pyruvate kinase deficiency].

Two children with nonspherocytic anaemia and pyruvate kinase deficiency had their diagnosis delayed due to normal initial estimations of enzyme activity. Repeated analyses involving other glycolytic enzymes documented an increased glucose-6-phosphate dehydrogenase activity as a function of high reticulocyte counts. The pyruvate kinase activity showed subnormal values. By simple comparison of both enzyme activities a true pyruvate kinase activity of below 50% was estimated thus rendering the diagnosis of pyruvate kinase deficiency highly probable. Analyses of more than one glycolytic enzyme should be performed in young children with otherwise unexplained haemolysis and associated high reticulocyte counts.

Anemia, Hemolytic, Congenital Nonspherocytic↗

PubMed1992

Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase.

The bifunctional reagent 1,4-dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min-1 M-1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme-bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 microM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit. Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn-Ile-X1-Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn-Ile-X1-Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn-Ile-X1-Lys cross-linked to X2-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross-linked peptides but not with formation of Asn-Ile-X1-Lys, inactivation is likely caused by the reaction leading to the cross-link between Cys151 and Cys164. The distance between the alpha-carbons of these residues in the crystal structure is 15.5 A, whereas only 12.0 A can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase undergoes a conformational change in forming the cross-link or that local rapid fluctuations in structure occur in solution to the extent of 3.5 A in this region of pyruvate kinase.

Amino Acid Sequence↗

PubMed1979

Functional changes associated with the sequential transformation of L'4 into L4 pyruvate kinase.

The functional changes, associated with the sequential transformation of L'4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L'4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the "final activity" (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP-or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase (either from human liver or trypsin treated L'4 enzyme) does not, or to a very slight extent, show such behaviour. L'2L2 pyruvate kinase shows behaviour intermediate between L'4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L'4 and L4 enzymes.

Adenosine Diphosphate↗

PubMed1998

Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth.

Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins--thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.

Amino Acid Sequence↗

PubMed2002

Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites.

We have cloned a cDNA encoding Toxoplasma gondii pyruvate kinase and obtained the full-length recombinant enzyme with a calculated molecular mass of 57.5 kDa. The predicted amino acid sequence of T. gondii pyruvate kinase exhibited a highest identity (63%) to that of Eimeria tenella pyruvate kinase and a lower identity of less than 25% to the pyruvate kinases from other organisms. Southern blot analysis indicated that the pyruvate kinase gene existed as a single copy in the T. gondii tachyzoite. The active recombinant enzyme contained four subunits and produced a strongly sigmoid saturation curve with phosphoenolpyruvate as the variable substrate. Fructose 1,6-diphosphate, a general activating factor of pyruvate kinase in most species, did not affect the enzyme activity. However, glucose 6-phosphate radically activated the enzyme. Fructose 2,6-diphosphate suppressed the reaction velocity at a higher concentration of phosphoenolpyruvate. These properties indicate that pyruvate kinase activity in T. gondii is regulated by unusual phosphorylated sugars.

Amino Acid Sequence↗

PubMed1979

Rat-brain pyruvate kinase: purification and effects of lithium.

Purified pyruvate kinase was prepared from pooled brains obtained from untreated rats. Its properties suggest that it is similar to type 'M' pyruvate kinase. Lithium inhibition was demonstrated at pharmacologically significant lithium concentrations (7%-12% at 2 mmol 1-1 Li) and this was similar in character to that previously seen in rabbit muscle pyruvate kinase, namely noncompetitive with respect to phosphoenol pyruvate, K+, and Mg2+ but competitive with ADP.

Animals↗

PubMed1981

Exogenous, but not endogenous, cyclic GMP reduces hepatic pyruvate kinase activity.

We investigated the effects of exogenous cyclic GMP and stimulants of endogenous cyclic GMP accumulation on L-form (hepatic) pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) activity in isolated rat hepatocytes. Exogenous cyclic GMP (200 muM) reduced pyruvate kinase activity, but was less potent than exogenous cyclic AMP (50 muM) (Ki congruent to 120 muM vs. 30 muM, respectively), had a slower onset of action (1.0 vs. 0.3 min, respectively) and a less rapid maximal effect (5.0 vs. 1.0 min, respectively). Similar results were noted with dibutyryl cyclic GMP or dibutyryl cyclic AMP. 1.0 muM acetylcholine increased cyclic GMP concentrations in isolated hepatocytes from 233 +/- 16 to 447 +/- 3 pmol/g cell protein (P less than 0.001), but did not alter pyruvate kinase activity. Similar results were noted with carbamylcholine, NaN3 or acetylcholine plus eserine sulfate. The results suggest a differential effect of exogenous vs. endogenous cyclic GMP on L-form pyruvate kinase activity, and question the physiological relevance of observations with exogenous cyclic GMP in this system.

Acetylcholine↗

PubMed1975

Investigation into pyruvate kinases from Escherichia coli K-12 grown under aerobic and anaerobic conditions.

Two forms of anaerobic Escherichia coli K-12 pyruvate kinase (EC 2.7.1.40) were separated by ammonium sulphate fractionations. Pyruvate kinases I is allosteric and pyruvate kinase II is non-allosteric to phosphoenolpyruvate. The addition of 1 mM FDP reversed the allostery to normal Michaelis-Menten kinetics. AMP had no effect, whereas 8 mM ATP completely inhibited the enzyme. The enzyme showed normal kinetics with ADP as substrate. Mg2+ and Mn2+ stimulated whereas Cu2+ severely inhibited the enzyme, which could be reversed by the addition of 1 mM FDP. Citrate, alpha-ketoglutarate, succinate, fumarate and alanine inhibited the enzyme, whereas phenylalanine had no effect. The allosteric pyruvate kinase from aerobic cultures was not only activated by FDP, but also by AMP. FDP changed Km and Vmax, whereas AMP influenced only the Km. During aerobic-anaerobic transition, pyruvate kinase synthesis increases and reaches a maximum under anaerobic conditions. The degree of FDP activation remains constant, but AMP activation is lost during transition. Aerobic cultures of E. coli K-12 grown on gluconeogenic substrates exhibited pyruvate kinase II activity (non-allosteric), which was stimulated by FDP and by AMP. It has been suggested that E. coli may have two types of pyruvate kinase II depending on the substrate and two types of pyruvate kinase I depending on oxygen tension in the medium.

Adenine Nucleotides↗