Presence Of Phosphoenolpyruvate Research Articles

Mechanosensitivity and mechanotransductive properties of osteoclasts.

Mechanical loading is essential for maintaining bone health. Here, we aimed to investigate the role of ATP and ADP in the mechanotransduction of bone-resorptive osteoclasts. Single osteoclast in primary cultures from 10 to 12-wk-old mice was mechanically stimulated by a gentle touch with a micropipette. Changes in cytosolic free calcium [Ca2+]i were analyzed in Fura-2 loaded osteoclasts. The cell injury was assessed by analyzing the cellular Fura-2 loss and classified as severe or mild using k-means. Osteoclasts responded to mechanical stimuli with transient calcium elevation (primary responders) and transduced these signals to neighboring cells, which responded with delayed calcium elevations (secondary responders). Severely injured osteoclasts had higher calcium transients than mildly injured cells. Fluid shear stress similarly induced reversible cell injury in osteoclasts. Secondary responses were abolished by treatment with A-804598, a specific inhibitor of P2X7, but not suramin, a broad P2 receptor blocker. Osteoclasts responded to ATP and ADP with concentration-dependent changes in [Ca2+]i. We performed osteoclast micropipette stimulation in the presence of phosphoenolpyruvate and pyruvate kinase which converted all ADP in solution to ATP, or with hexokinase converting all ATP to ADP. Osteoclasts with mild membrane injury demonstrated similar calcium responses in ATP and ADP-rich environments. However, when the mechanotransductive signal to severe osteoclast injury was converted to ADP, the fraction of secondary responders and their [Ca2+]i amplitude was higher. This study suggests the importance of osteoclast mechanobiology and the role of ADP-mediated signaling in conditions of altered mechanical loading associated with bone loss.NEW & NOTEWORTHY Osteoclasts are rarely considered as cells that participate in mechanical signaling in bone. We show that osteoclasts are capable of sensing and transmitting mechanical signals to neighboring cells. Mechanical stimulation commonly induces minor repairable membrane injury in osteoclasts. ATP and especially ADP were found to play important roles in the mechanoresponsiveness of osteoclasts. This study highlights the importance of osteoclast mechanobiology especially in conditions of altered mechanical loading associated with bone loss, such as in microgravity.

Structure‐function studies of ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii: Probing the Role of the C‐terminus in Allosteric Regulation

Starch serves as both a critical food source and an important biodegradable and renewable carbon source for biofuel and other industries including adhesives, packaging material, and cosmetics. ADP‐glucose pyrophosphorylase (ADPG PPase, glgC gene product) is the key enzyme that is responsible for the rate‐limiting step of both glycogen and starch biosynthesis in bacteria and plants, catalyzing the conversion of glucose‐1‐phosphate and ATP to ADPG and pyrophosphate. Thermodesulfovibrio yellowstonii (Td. y) is a thermophilic sulfate‐reducing bacterium that was isolated from geothermal vents in Yellowstone National Park. Little is known about this ADPG PPase which displays increased heat stability, salt tolerance, and altered regulation compared to other ADPG PPase family members. The Td.y enzyme is substantially activated by phosphoenolpyruvate (PEP), which differs from many other ADPG PPases, as well as G6P, 3PGA, and sulfate. These properties could contribute to biomass production in different crop environments. Multiple sequence alignments have shown deviations from consensus residues in the N‐terminal allosteric region and C‐terminal region of the Td.y ADPG PPase. Two of the more interesting differences are at positions 365 and 397, which have been shown to be involved in allosteric regulation of other ADPG PPases, as part of a conserved region in the enzyme family and associated with plant enzymes, respectively. The K365G and R397A enzymes were generated by site‐directed mutagenesis as a first step in probing structure‐function relationships. We hypothesize that the positively charged lysine at 365 and arginine at 397 may contribute to PEP binding and activation. The altered proteins were purified to homogeneity by a procedure that included two hydroxyapatite chromatography steps followed by size exclusion chromatography. Initial kinetic studies revealed that the K365G protein displayed a lower Vmax (~25% of wild‐type [WT]) but ~2‐fold higher apparent affinity for ATP in the absence of effectors. In contrast to the WT, which was activated over 3‐fold by PEP, the K365G enzyme only exhibited ~10% activation by PEP and a decrease in apparent affinity for ATP. Conversely, the K365G enzyme was activated 2.6 fold by sulfate, an effector which has a much smaller effect on the Vmax of WT. In the absence of effectors, the R397A displayed a similar apparent affinity for ATP compared to WT but a 24 fold decrease in Vmax. In the presence of PEP, R397A exhibited lower apparent affinity for ATP and a Vmax decreased ~42 fold. The response of this enzyme to G6P was very similar to WT. The results so far support the hypothesis that K365 and R397 are critical for PEP activation while also influencing other allosteric properties. Complete kinetic analyses of the K365G and R397A enzymes as well as crystallization trials are underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

Background: The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1].Objective: Provide the first biochemical and biophysical characterization of the allelic variant G233D of TIM.Methods: The Michaelis-Menten curves using both substrates of TIM were obtained. Also the effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed in presence of GAP and DHAP. The thermal stability in absence and presence of PEP was analyzed by circular dichroism spectroscopy. For comparison purposes, all the measurements were carried out on the wild type TIM and variant G233D.Results: The G233D variant exhibited a kcat value 4-fold lower than that of the WT enzyme in the GAP isomerization to DHAP, which is the reverse reaction of the glycolytic pathway. The G233D variant exhibited Ki and IC50 values of 120 μM and 356 μM in the presence of several concentrations of GAP and 0.3 mM DHAP, respectively. These inhibition parameters are similar to those exhibited by the WT enzyme. The thermal unfolding cooperativity of G233D variant was significantly increased upon PEP binding, suggesting that the ligand-bound enzyme was trapped in a rigid conformation.Conclusion: We suggest that the flow of GAP through glycolysis could be enhanced by the decreased activity of the G233D variant in the formation of DHAP.

ENZYME ACTIVITIES INVOLVED IN ATP SYNTHESIS IN SEEDS

Cell-free extracts from seeds of onion (Allium cepa), peas (Pisum sativum) and lettuce (Lactuca sativa) exhibit ATP synthesis in the presence of phosphoenolpyruvate (PEP) and AMP. The activity is stimulated by low amounts of ADP or ATP. Reduced NAD replaced AMP in some seeds, and increased the rate of ATP synthesis in the presence of AMP in others. If ADP and PEP were used as substrates (pyruvate kinase), a 5- to 50- times faster ATP synthesis was observed. Examination of enzymes from pea and onion seed extracts revealed that three different enzymes are involved in the conversion of AMP to ATP: (a) pyruvate kinase (EC 2.7.1.40), (b) adenylate kinase (EC 2.7.4.3) and (c) an activity which results in formation of ADP from reduced NAD. The overall activity for ATP synthesis from AMP is stimulated by K+, Mg2+, Mn2+ and Co+, and is inhibited by Na+ and Ca2+. A scheme for the possible pathway of ATP synthesis in seeds at the early stage of germination is presented.

Phosphoenolpyruvate: Sugar Phosphotransferase System from the Hyperthermophilic Thermoanaerobacter tengcongensis

Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ∼90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 Å resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 Å crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 Å away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.

Synaptic Vesicle-bound Pyruvate Kinase can Support Vesicular Glutamate Uptake

Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [(3)H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. "ATP-trapping" experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.

Biophysical Characterization of the Enzyme I of the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System

The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg 2+. The conformational stability and structure of the EI from Streptomyces coelicolor, EI sc, were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI sc was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI sc had a well-folded structure; however, at low pH, EI sc showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI sc, in the absence of PEP and Mg 2+, was maximal at pH 7. The presence of PEP and Mg 2+ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI sc was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.

Crystal Structure of the Reaction Complex of 3-Deoxy-d-arabino-heptulosonate-7-phosphate Synthase from Thermotoga maritima Refines the Catalytic Mechanism and Indicates a New Mechanism of Allosteric Regulation

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPSTm) were grown in the presence of PEP and metal cofactor, Cd2+, and then soaked with E4P at 4 °C where the catalytic activity of the enzyme is negligible. The crystal structure of the “frozen” reaction complex was determined at 2.2Å resolution. The subunit of the DAHPSTm homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (β/α)8-barrel domain. The active site located at the C-end of the barrel contains Cd2+, PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPSTm is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPSEc) and Saccharomyces cerevisiae (DAHPSSc). However, the secondary, tertiary, and quaternary structures of DAHPSTm are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPSEc and DAHPSSc. Although DAHPSTm is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPSEc and DAHPSSc. A sequence similarity search revealed that DAHPSs of phylogenetic family Iβ possess a FL domain like DAHPSTm while those of family Iα have extra barrel segments similar to those of DAHPSEc and DAHPSSc. This indicates that the mechanism of feedback regulation in DAHPSTm and other family Iβ enzymes is different from that of family Iα enzymes, most likely being mediated by the FL domain.

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.

Activation of 6-phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

Metal-Catalyzed Oxidation of Phenylalanine-Sensitive 3-Deoxy- d - arabino -Heptulosonate-7-Phosphate Synthase from Escherichia coli : Inactivation and Destabilization by Oxidation of Active-Site Cysteines

The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.

Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP‐dependent, enzyme I‐ and HPr‐catalysed phosphorylation

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.

An Alternative and Inexpensive Assay for Plant Invertases

The glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose oxidase methods are commonly adapted for plant invertase assay. A disadvantage of the G-6-PDH assay is the relatively high cost of the coupling enzymes and cofactors. A disadvantage of the glucose oxidase method, which uses a glucose kit (Sigma, 510-A), is the presence of high activities of acid invertase and alkaline invertase in the PGO enzyme formula (peroxidase and glucose oxidase), which gives a falsely high invertase activity value. An alternative and inexpensive coupled assay was developed for enzymatic assay of plant invertases. In this assay, ADP produced from phosphorylation of glucose and fructose (hydrolysis products of invertases) is coupled to oxidation of NADH by the enzymes pyruvate kinase and lactate dehydrogenase in presence of phosphoenolpyruvate and NADH. This method was compared with the glucose-6-phosphate dehydrogenase method by using protein preparations derived from plant materials of three different species. Statistical analysis indicated that the alternative assay was similar in accuracy to the glucose-6-phosphate dehydrogenase method, with an advantage of reducing the cost from $0.85 to $0.35 per assay.

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

Interaction studies between elongation factor Tu and anthraniloyl-fluorescent analogues of guanyl nucleotides.

A fluorescent analogue of GDP, the 3'-O-anthraniloyl-GDP (anl-GDP) was demonstrated to bind to the elongation factor Tu (EF-Tu) with an affinity even higher than that of the parent nucleotide. As a consequence of the binding, an increase in fluorescence anisotropy and an emission band arising from non-radiative energy transfer among the protein intrinsic fluorophores and the labelled nucleotide were observed. Therefore, it was possible to study the exchange kinetics and the equilibrium between the protein-bound labelled GDP and the natural nucleotide through modifications, occurring during the course of the reaction, of fluorescence anisotropy and non-radiative energy transfer. In this way, it was also easily proven that, in the presence of aurodox (N-methylkirromycin), an antibiotic impairing EF-Tu biological function, the exchange kinetics between the protein-bound labeled GDP and the natural nucleotide was faster. Moreover, it was also found that the labelled nucleotide is recognized as a substrate by pyruvate kinase, being converted by this enzyme, in the presence of phosphoenolpyruvate, into anl-GTP. Pyruvate kinase is also able to convert, in the presence of phosphoenolpyruvate, the complex EF-Tu.anl-GDP into the complex EF-Tu.anl-GTP. The fluorescence properties of the 3'-O-anthraniloyl-labeled guanyl nucleotides and their feature as excellent acceptors of fluorescence arising from protein intrinsic fluorophores, may make these compounds useful for structural and binding studies on guanosine-nucleotide-binding proteins.

Evidence for a phosphoenolpyruvate dependent sugar-phosphotransferase system in the mollicute Acholeplasma florum

In order to confirm the presence of a phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS) in the mollicute Acholeplasma florum we studied the ability of cell free extracts of this organism to phosphorylate glucose and/or fructose in the presence of PEP. We also cloned and sequenced a DNA fragment coding for a putative polypeptide showing significant similarity with the enzyme II of the ß-glucoside PTS of Escherichia coli. Taken together, these results show that A florum possesses a fructose-PTS, but not a glucose-PTS, and that the amino acid sequence deduced from the DNA fragment is related to ß-glucoside and sucrose enzymes II of PTS from various bacteria.

Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K(m), and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.

Purification and properties of sorbitol-6-phosphate dehydrogenase from oral streptococci

The activity of sorbitol-6-phosphate (S6P) dehydrogenase (S6PDH) and the sorbitol transport system were studied in strains of the oral streptococci Streptococcus gordonii, Streptococcus mitis, Streptococcus sanguis and Streptococcus mutans. Genetically transformed (to ferment sorbitol) strains and their DNA donors were included. S6PDH was purified by anion exchange chromatography and gel filtration. The purity of the enzyme was confirmed by polyacrylamide gel electrophoresis. The purified enzyme from all the strains exhibited Michaelis-Menton saturation kinetics. The Km values for nicotinamide-adenine dinucleotide (NAD) and S6P ranged between 0.03 and 0.21 mM and 0.07 and 0.20 mM respectively. The relative molecular weights of the native enzyme were 229,000 for one donor-transformant pair (S. sanguis and S. gordonii), 107,000 for the other pair (S. mitis and S. gordonii) and 129,000 for S. mutans. The molecular weights of the S6PDH subunits ranged from 26,000 to 28,000. The pH optima (greater than 8.5) and the amino acid composition (15 amino acids examined) were similar for the S6PDH from the different strains. However, the chromatographic and electrophoretic patterns as well as the Km values for NAD and S6P were the same only between the S6PDHs from the strains within each donor-transformant pair. Purified S6PDH from S. mutans also exhibited low mannitol-1-phosphate dehydrogenase activity. Sorbitol-grown decryptified cells of all the strains phosphorylated sorbitol in the presence of phosphoenolpyruvate but not in the presence of adenosine triphosphate (ATP). ATP-mediated phosphorylation of glucose was observed with the same strains when grown on glucose. No evidence for a non-phosphotransferase transport system was found for sorbitol in any of the strains.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effect of growth conditions on the Streptococcus bovis phosphoenolpyruvate glucose phosphotransferase system1

Four strains of the ruminal bacterium Streptococcus bovis were surveyed for phosphoenolpyruvate (PEP) and ATP-dependent phosphorylation of glucose and the nonmetabolizable glucose analog 2-deoxyglucose. All four strains had high rates of glucose phosphorylation with either phosphoryl donor, but 2-deoxyglucose activity was much higher in the presence of PEP. These results provide evidence for a PEP-dependent glucose phosphotransferase system in these bacteria. Mannose and 2-deoxyglucose inhibited PEP-dependent phosphorylation of glucose by S. bovis JB1 by 50 and 38%, respectively, whereas alpha-methylglucoside had little effect. Mannose was a competitive inhibitor of PEP-dependent phosphorylation of glucose with an inhibition constant of 2.8 mM, and PEP-dependent activity in cells grown in batch culture was optimal at pH 7.2. When S. bovis JB1 was grown in continuous culture, PEP-dependent phosphorylation of glucose and 2-deoxyglucose was highest in cells grown at a dilution rate of .10/h and at low glucose concentrations. Phosphoenolpyruvate-dependent activity was optimum at a growth pH of 5.0 for cells grown in medium that contained less than 6.0 g/liter of glucose. These data indicate that PEP-dependent glucose phosphotransferase system activity can be influenced depending on the growth conditions used to culture S. bovis. Furthermore, these results suggest that environmental conditions within the rumen will affect how glucose is transported by S. bovis.