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The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

The prolyl hydroxylase 3 (PHD3) protein is less abundant in normal oxygen conditions (normoxia) but increases under deficient oxygen condition (hypoxia). Since cancerous cells often thrive in hypoxic conditions and predominantly express the Pyruvate kinase isoforms 2 (PKM2), the PHD3/PKM2 interaction might be particularly important in cancer development. In the present study, the PHD3/PKM2 complex was co-expressed and purified by size-exclusion chromatography. The interaction of PHD3 with PKM2 was confirmed in Native gel as well as western blot analysis. The PHD3/PKM2 complex formed discreet crystals under suitable conditions, and diffraction data revealed that crystal belonged to the P1 space group with 3.0 Å resolution. This is the first crystal report of PHD3/PKM2 complex as well as this study demonstrates a direct physical binding through protein-protein interaction. The structural analysis of complex will provide the information regarding the amino acid residues critical for the catalytic mechanism. Based on the structural information thus obtained, pharmacological interference with the PHD3/PKM2 interaction could be used as a novel strategy to reduce the cancer progression.

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Ground squirrel torpor during winter hibernation is characterized by numerous physiological and biochemical changes, including alterations to fuel metabolism. During torpor, many tissues switch from carbohydrate to lipid catabolism, often by regulating key enzymes within glycolytic and lipolytic pathways. This study investigates the potential regulation of pyruvate kinase (PK), a key member of the glycolytic pathway, within the skeletal muscle of hibernating ground squirrels. PK was purified from the skeletal muscle of control and torpid Richardson's ground squirrels, and PK kinetics, structural stability, and posttranslational modifications were subsequently assessed. Torpid PK displayed a nearly threefold increase in K m PEP as compared to control PK when assayed at 5 °C. ProQ Diamond phosphoprotein staining as well as phospho-specific western blots indicated that torpid PK was significantly more phosphorylated than the euthermic control. PK from the torpid condition was also shown to possess nearly twofold acetyl content as compared to control PK. In conclusion, skeletal muscle PK from the Richardson's ground squirrel may be regulated posttranslationally between the euthermic and torpid states, and this may inhibit PK functioning during torpor in accordance with the decrease in glycolytic rate during dormancy.

Background: Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research., Results: Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro., Conclusions: The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

Infrared spectroscopy is a powerful tool in protein science due to its sensitivity to changes in secondary structure or conformation. In order to take advantage of the full power of infrared spectroscopy in structural studies of proteins, complex band contours, such as the amide I band, have to be decomposed into their main component bands, a process referred to as curve fitting. In this paper, we report on an improved curve fitting approach in which absorption spectra and second derivative spectra are fitted simultaneously. Our approach, which we name co-fitting, leads to a more reliable modelling of the experimental data because it uses more spectral information than the standard approach of fitting only the absorption spectrum. It also avoids that the fitting routine becomes trapped in local minima. We have tested the proposed approach using infrared absorption spectra of three mixed α/β proteins with different degrees of spectral overlap in the amide I region: ribonuclease A, pyruvate kinase, and aconitase.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni(2 +) chelation chromatography yielded a single protein with an apparent molecular mass of 58kDa and a specific activity of 34unitsmg(-1) protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2min at 75°C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas α-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37°C and in the pH range from 7.4 to 7.8. K(M) constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mM and for PEP to 0.07±0.01 mM. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95%). The metal ions Mg(2+) and K(+) are necessary for full enzymatic activity. The addition of transition metal ions such as Mn(2+), Cu(2+), Co(2+), and Ni(2+) reduces activity. Storage of the enzyme at -20°C retains full activity.

The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Pyruvate kinase (PK) is the key control point of glycolysis-the biochemical pathway central to energy metabolism and the production of precursors used in biosynthesis. PK type 1 from Escherichia coli (Ec-PK1) is activated by both fructose-1,6-bisphosphate (FBP) and its substrate, phosphoenol pyruvate (PEP). To date, it has not been possible to determine whether the enzyme is tetrameric at the low concentrations (i.e. low nM range) used to study the steady-state kinetics, or assess whether its allosteric effectors alter the oligomeric state of the enzyme at these concentrations. Employing the new technique of analytical ultracentrifugation with fluorescence detection we have, for the first time, shown that the K(D)(4-2) for Ec-PK1 is in the subnanomolar range, well below the concentrations used in kinetic studies. In addition, we show that, unlike some other PK isoenzymes, the modulation of oligomeric state by the allosteric effectors FBP and PEP does not occur at a concentration of 10 nM or above., (2009 Elsevier Masson SAS. All rights reserved.)

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.

Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation.

Antibodies against Brassica napus cytosolic pyruvate kinase (PKc) (EC 2.7.1.40) were employed to examine PKc subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PKc activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (micromol of pyruvate produced/min) g(-1) FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PKc activity and the relative amount of the immunoreactive 56-kDa PKc polypeptide. PKc from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (micromol of pyruvate produced/min) mg(-1) protein. Gel filtration and SDS-PAGE indicated that this PKc exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PKc subunit best matched three putative PK(c)s from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of Vmax /Km for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PKc exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.

Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.

Cryptosporidium parvum is one of the major causes of waterborne diseases worldwide. This protozoan parasite depends mainly on the anaerobic oxidation of glucose for energy production. In order to identify the differences in the three-dimensional structure of key glycolytic enzymes of C. parvum and its human host, we have expressed, purified and crystallized recombinant versions of three important glycolytic enzymes of the parasite, namely, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. Lactate dehydrogenase has been crystallized in the absence and in the presence of its substrates and cofactors, while pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase were crystallized only in the apo-form. X-ray diffraction data have been collected for all crystals.

The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine. T298C YPK was isolated and purified, and its enzymatic properties were characterized. Fluorescence and CD spectra indicate minor structural perturbations. A kinetic analysis of the Mg(2+)-activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP). In the presence of Mg(2+) and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme. T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n(H,FBP) = 1). Mn(2+)-activated T298C behaves like Mn(2+)-activated wt YPK with a V(max) that is 20% of that for the wt enzyme with or without FBP. A pH-rate profile of T298C relative to that for wt YPK shows that pK(a,2) has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK. A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg(2+) complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of FBP to all the relevant complexes. Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wt and T298C. Proton inventory studies indicate that k(cat) is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.

Pyruvate kinase is the final regulatory point in the catabolic Embden-Meyerhoff-Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. trachomatis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis-Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was allosterically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.

Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved.

Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein. The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits. This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs. The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803. Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0. Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate. Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators. The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.

To elucidate the regulatory mechanism for propionate production in Selenomonas ruminantium, the molecular properties and gene expression of phosphoenolpyruvate carboxykinase (Pck) and pyruvate kinase (Pyk) were investigated. The Pck was deduced to consist of 538 aa with a molecular mass of 59.6 kDa, and appeared to exist as a monomer. The Pyk was revealed to consist of four identical subunits consisting of 469 aa with a molecular mass of 51.3 kDa. Both Mg(2+) and Mn(2+) were required for the maximal activity of Pck, and Pck utilized ADP, not GDP or IDP, as a substrate. Either Mg(2+) or Mn(2+) was required for Pyk activity, and the enzyme was activated by phosphoenolpyruvate (PEP) and fructose 1,6-bisphosphate (FBP). Pyk activity was severely inhibited by P(i), but restored by the addition of FBP. The K:(m) value of Pck for PEP (0.55 mM) was nearly equal to the K:(m) value of Pyk for PEP, suggesting that the partition of the flow from PEP in the fermentation pathways is determined by the activity ratio of Pck to Pyk. Both pck and pyk genes were monocistronic, although two transcriptional start sites were found in pyk. The level of pyk mRNA was not different whether glucose or lactate was the energy substrate. However, the pck mRNA level was 12-fold higher when grown on lactate than on glucose. The level of pck mRNA was inversely related to the sufficiency of energy, suggesting that Pck synthesis is regulated at the transcriptional level when energy supply is altered. It was conceivable that the transcription of pck in S. ruminantium is triggered by PEP and suppressed by ATP.

Pyruvate kinase from skeletal muscle of lamprey (Entosphenus japonicus), which is one of the most primitive living vertebrates, has been purified by approxImately 110-fold. The isolation procedure includes chromatography on Phosphocellulose, Phenyl-5PW, and Sephacryl S-300. Sodium dodecyl sulfate gel electrophoresis shows 59000 as the deduced subunit molecular weight and gel filtration shows 232000 as the tetramer of the subunits. The apparent Km for phosphoenolpyruvate and ADP are 0.41 mM and 0.31 mM at pH 7.4, respectively, when the purified enzyme is saturated with the second substrate. When the enzyme is activated in the presence of fructose-1,6-diphosphate, the Km for PEP changes to 0.087 mM, and the Hill coefficient changes from 1.3 to 0.98.

Cytosolic pyruvate kinase (PK(c)) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7 micromol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240 kDa homotetramer composed of subunits of 57 kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of V(max)/K(m) for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH 6.9 and 7.5. PK(c) activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg(2+) and K(+) respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg(2+) and K(+) (K(m) values of 0.098, 0.12, 0.27 and 0.91 mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PK(c) by L-glutamate. The allosteric features of banana PK(c) are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807-816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

The specific activity and the kinetic properties of partly purified pyruvate kinase (PK) (EC 2.7.1.40) from the Northern Krill, Meganyctiphanes norvegica, were investigated in relation to varying food resources. In order to evaluate the effect of starvation on the total energy metabolism, the respiration rates of fed and unfed krill were determined. The FPLC-elution profile of PK displayed two distinct peaks - PK I and II. The first isoform represented 80% of the total PK activity in the organism, and 20% was contributed by the second isoform. PK I was inhibited by ATP but was not influenced by fructose-1,6-bisphosphate (FBP). In contrast, PK II showed ATP inhibition and up to 2.5-fold increased activity by addition of 17 micromol.l(-1) FBP. The Michaelis-Menten constants of both isoforms were 2-10-fold higher for ADP than for phosphoenolpyruvate (PEP). Alanine showed no regulatory effect on PK I and II. In specimens starved for 7 days oxygen consumption decreased by 20%. Neither the feeding experiments nor the animals captured in the field during low and high productive seasons indicate that PK properties of M. norvegica are modified in relation to food supply. Accordingly, alternative mechanisms are involved in the depression of the metabolic rate in terms of oxygen consumption.

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively). L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.

We describe the isolation and characterization of a pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius. This protein appears to be a tetramer composed of four 55-kDa subunits. The intrinsic tryptophan fluorescence of this protein is quenched by approximately 20% upon binding sodium, which occurs with a dissociation constant near 15 mM. Importantly, the intrinsic fluorescence of this pyruvate kinase does not appear to be affected by potassium, magnesium, and calcium at the concentrations found in whole blood. It appears that this pyruvate kinase can provide the basis for a selective protein sensor for sodium with minimal interference from other cations.

Pyruvate kinase (PK; EC 2.7.1.40) of Thermoproteus tenax was purified to homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg(2+) and Mn(2+). Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K(+) ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)-Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris-HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)-Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)-Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)-Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.

Cytosolic pyruvate kinase (ATP: Pyruvate phosphotransferase, EC 2.7.1.40; PKc) was purified to apparent homogeneity with about 22% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive Blue Sepharose-CL-6B. The purified enzyme with molecular mass of about 214 kDa was a heterotetramer with subunit molecular mass of 55 and 57 kDa. The enzyme showed maximum activity at pH 6.8 and absolute requirement for a divalent (Mg2+) and a monovalent (K+) cation for activity. Typical Michaelis-Menten kinetics was obtained for both the substrates with Km values of 0.10 and 0.11 mM for PEP and ADP, respectively. The enzyme could also use UDP or GDP as alternative nucleotides, but with lower Vmax and lesser affinities. The enzyme was inhibited by glutamate, glutamine, fumarate, citrate, isocitrate, oxalate, 2-PGA, ATP, UTP and GTP and activated by glucose-6-phosphate, fructose-1,6-bisphosphate and Pi, suggesting its regulation mainly by TCA cycle intermediates and the cellular need for carbon skeletons for amino acid biosynthesis. ATP inhibition was of competitive type with respect to PEP and non-competitive with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. Initial velocity and product inhibition studies except for pyruvate inhibition were consistent for a compulsory-ordered tri-bi mechanism.

Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively. Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK. The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK. The detritiation of 3-[(3)H]pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively. This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M. Isotope trapping was used to characterize enolpyruvate protonation by K240M. While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M. In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8. 8 observed with wild-type YPK. The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.

A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.

Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and non-competitively with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.

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., (Copyright 1998 Academic Press.)

Chromatography on immobilized antibodies specific to nucleoside diphosphate (NDP) kinase was utilized for affinity purification of this enzyme from detergent extracts of frog heart post-mitochondrial fractions. SDS-polyacrylamide gel electrophoresis analysis of eluates from these supports shows that five polypeptides co-purify with nucleoside diphosphate (NDP) kinase. Tryptic digests of each band were analyzed by mass spectrometric microsequencing. Data base searches by peptide mass matching and sequence homology led to the identification of these proteins as glyceraldehyde-3-phosphate dehydrogenase (40 kDa), creatine kinase (45 kDa), vimentin (55 kDa), pyruvate kinase (60 kDa), and a putative member of the antioxidant protein family (28 kDa). Distinct protein compositions were found in eluates of lung and liver extracts processed in a like manner. The 28-kDa band and vimentin were associated with NDP kinase from all tissues, but co-purification of pyruvate kinase was seen only in liver, while creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were absent from eluates from lung and liver. The results suggest that while NDP kinase is associated with vimentin intermediate filaments and an antioxidant protein in most tissues, it interacts with energy metabolism enzymes in a tissue-specific manner.

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.

Cytosolic pyruvate kinase (PKc) from leaves of the castor oil plant (Ricinus communis L.) has been purified 3900-fold to apparent homogeneity and a final specific activity of 51 mumol of pyruvate produced/min/mg protein. PAGE, immunoblot, and gel filtration analyses of the final preparation indicated that this enzyme is an alpha 2 beta 2 heterotetramer of about 250 kDa that is composed of an equivalent ratio of 57- and 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of approximately 6.5. However, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] occurred at pH 7.5. Enzyme activity was absolutely dependent upon the simultaneous presence of bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed with PEP, ADP, and K+, whereas Mg2+ binding exhibited positive cooperativity. Mg2 citrate, oxalate, and glutamate were the most effective inhibitors at pH 7.5. Inhibition by these compounds was more pronounced at pH 7.5 than at pH 6.5 and they yielded additive inhibition when tested in pairs. Aspartate functioned as an activator by facilitating the binding of PEP and relieving the inhibition of PKc by glutamate. The in vivo activity of leaf PKc is probably regulated by the relative cytosolic levels of citrate, glutamate, and aspartate. This provides a possible rationale for the known activation of leaf PKc that occurs during periods of enhanced ammonia assimilation. Together with our previous studies, the results also indicate that castor oil plant PKc exists as tissue-specific isoforms that demonstrate substantial differences in their respective physical and/or kinetic and regulatory properties.

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant, Km, for phosphoenol pyruvate and a more than ten times higher Kcat/Km value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower Km value and a 100-fold increased kcat/Km value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

A practical study is presented of the influence of cell debris and polymer recycling upon the operation of two-stage aqueous two-phase systems (ATPS) for the recovery of yeast bulk protein, pyruvate kinase and fumarase. Brewers' yeast was disrupted using one of two types of high-pressure homogenisers or a bead mill. The different cell debris suspensions were partitioned in a single PEG-phosphate ATPS extraction and the efficiency of solid-liquid separation was examined. A continuously operated two-stage ATPS process, using spray columns, is presented and practical problems of polymer recycling are discussed. Conclusions are drawn concerning the generic implementation and operational stability of ATPS in practical protein recoveries.

Oocysts of Cryptosporidium parvum were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) similar to those previously described for Eimeria tenella and Toxoplasma gondii. PPi-PFK of C. parvum displayed simple hyperbolic kinetics with respect to its substrate fructose 6-phosphate and was not affected by fructose 2,6-bisphosphate, the major allosteric activator of most ATP-PFKs. Inorganic pyrophosphatase was not detectable in any of the three parasites. T. gondii tachyzoites and C. parvum cysts both contained a pyruvate kinase (PK) specific for ADP rather than PPi/AMP. The PK of T. gondii was similar to that of E. tenella in that it displayed strong positive cooperativity with respect to its substrate phosphoenolpyruvate and was heterotropically activated by glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. PK of C. parvum showed no evidence of allosteric properties. The results suggest that the three coccidia are similar in depending heavily on anaerobic energy production via glycolysis but that the mechanisms for regulating glycolysis are not common to all species.

Cytosolic pyruvate kinase fractions A and B obtained by salting out procedure from normal rat liver and Morris hepatoma 7777, purified by affinity chromatography on Blue Sepharose CL-6B, have shown similar electrophoretic patterns in polyacrylamide gel at pH 8.3 to previously studied pyruvate kinase extracts from chromatin of cell nuclei. Three variants (alpha 1, beta 1, gamma 1) from normal rat liver pyruvate kinase fraction A (type L) had the greatest electrophoretic mobility, showed sigmoidal kinetics in relation to 2-phosphoenolpyruvate (PEP), and sensitivity to ATP and fructose 1,6-diphosphate (FDP). The fraction A dominated over normal liver fraction B (type M2), which in electrophoresis showed a slower gamma 2 variant, similar to the fraction A of hepatoma. All variants from fractions B of normal liver and A of hepatoma had linear kinetics and were sensitive to ATP but not to FDP. The greatest differences showed pyruvate kinase fraction B from Morris hepatoma. Its all variants alpha 2, beta 2, gamma 3 were cathodic and had linear kinetics in relation to PEP. They all were insensitive to normal signal molecules (ATP and FDP). The gamma 3 alkaline variant acquired sensitivity to inhibition by L-cysteine. Showing several-fold higher activity, much greater affinity to the main substrate, and a lack of sensitivity to feed-back inhibition by ATP, it was responsible for a high rate of aerobic glycolysis and diminution of the Pasteur effect in metabolic studies. It was probably encoded during oncogene activation and plays a special role in different metabolic strategies of tumour cells.

There are four pyruvate kinase isozymes in vertebrate tissues, designated as L, M1, M2, and R. Although pyruvate kinases have been purified and characterized from pig liver, muscle, kidney, and heart, the brain isozyme has not. The aim of this work was to purify, characterize and make an isozymic designation for the pig brain pyruvate kinase. Purification was accomplished by chromatography on phosphocellulose, Sephadex G200, and blue-dextran agarose columns. The molecular weight of the native enzyme was determined by sucrose density centrifugation. The degree of purity, and subunit molecular weight were determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid isoelectric focusing method in sucrose gradients. The pH optimum, and kinetics in the presence and absence of fructose-1,6-diphosphate were determined spectrophotometrically. The purification scheme used resulted in a 382-fold purification of pig brain pyruvate kinase, and a final specific activity of 191 Units/mg protein. As estimated by scanning of the sodium dodecyl sulfate polyacrylamide gels, the purification scheme also resulted in a preparation that was of at least 98% purity. Pig brain pyruvate kinase has a native molecular weight of approx. 230,000, and a subunit molecular weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimum was estimated at pH 7.4. Fructose-1,6-diphosphate had no effect on either the Km for phospho(enol)pyruvate, or the Vmax of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.

The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.