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PubMed1987

Sugar transport by the bacterial phosphotransferase system. The intrinsic fluorescence of enzyme I.

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system has 2 tryptophan residues/monomer, as determined spectrophotometrically. The tryptophan fluorescence has been investigated with the aid of nanosecond time-resolved techniques. The decay of the fluorescence intensity was analyzed in terms of a biexponential function. The contribution of the emission associated with the shorter decay constant increases from 17-19% at 1 degree C to 43-44% at room temperature. Decay-associated spectra obtained with Enzyme I indicate different spectral distributions associated with the two decay constants. The measurement of tumbling of Enzyme I as a function of temperature revealed a transition of rotational rates between 5 and 15.5 degrees C. Global analysis allowed decomposition of the anisotropy decay into a formulation consistent with monomer and dimer rotational contributions.

Escherichia coli↗

PubMed1987

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles.

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.

Carbohydrate Metabolism↗

PubMed1986

Limited proteolysis of IIIGlc, a regulatory protein of the phosphoenolpyruvate:glycose phosphotransferase system, by membrane-associated enzymes from Salmonella typhimurium and Escherichia coli.

In the present studies we report that membrane-associated proteases in Salmonella typhimurium and Escherichia coli catalyze limited proteolysis of IIIGlcSlow. We have previously reported (Meadow, N. D., and Roseman, S. (1982) J. Biol. Chem. 257, 14526-14537) the isolation of two electrophoretically distinguishable forms of IIIGlc, which is a phosphocarrier and regulatory protein of the phosphoenolpyruvate:glycose phosphotransferase system. The two species of IIIGlc were designated IIIGlcFast and IIIGlcSlow; IIIGlcSlow is 7 amino acid residues longer than IIIGlcFast at its NH2 terminus. The majority of the protease activity is located in the outer membrane fraction from both species of bacteria, with the cytoplasmic fraction being devoid of activity. The site of cleavage is at the Lys-Ser bond located at residues 7-8 of IIIGlcSlow. The enzyme is an endopeptidase which liberates the expected heptapeptide (Gly-Leu-Phe-Asp-Lys-Leu-Lys). Both the large fragment of the limited proteolytic reaction, IIIGlcFast, and the small fragment, the heptapeptide, are stable to further proteolysis by membranes for more than 17 h at 37 degrees C. The activity in E. coli membranes has an absolute requirement for divalent metal ion (Mg2+ or Ca2+) and is heat-resistant, whereas the activity in S. typhimurium membranes is stimulated by divalent metal ion and is heat-sensitive. These results suggest significant differences between the two enzymes. The physiological function of the limited proteolysis of IIIGlc is not known.

Cations, Divalent↗

PubMed1987

Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon.

The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene.

Amino Acid Sequence↗

PubMed1978

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.

Biological Transport↗

PubMed1985

The phosphoenolpyruvate-dependent phosphotransferase system: a central feature of carbohydrate accumulation by enteric bacteria.

Despite the fact that bacterial solute transport has been intensively studied for the last two decades, it has not yet been possible to describe the molecular mechanism of any transport process, largely due to the difficulties involved in handling membrane proteins and the lack of convenient in vitro assay procedures. The phosphoenolpyruvate: glycose phosphotransferase, a multiprotein system consisting of both soluble and membrane-bound components and with an associated chemical reaction which can be measured in cell-free extracts, has considerable potential for attaining this goal. Although the PTS is extremely complex, our understanding of the mechanisms by which it mediates and regulates carbohydrate transport in enteric bacteria is at an advanced stage.

Carbohydrate Metabolism↗

PubMed1977

[Isolation and substrate specificity of neomycin (paromomycin)--phosphotransferase from Actinomyces fradiae, a producer of neomycin].

Neomycin (paromomycin) phosphotransferase was isolated from the mycelium and fermentation broth filtrates of Act. fradiae. The substance was partially purified by means of fractionation with ammonium sulphate followed by gel-filtration through Sefadex G-100. The extracellular and intracellular forms of the enzyme had the same substrate specificity and used only neomycin and paromomycin as substrates. The other aminoglycosides, including kanamycins A and B, lividomycin and ribostamycin were not used. The both forms had the same thermolability. The intracellular form of the enzyme was detected in the mycelium at the early stages of the organism development, while the extracellular form was found in detectable amounts in the culture medium only at the late stages of the actinomycete development. Therefore, the neomycin-producing organism, i.e. Act. fradiae had one enzyme which phosphorilated neomycin and paromomycin and was excreted from the mycellium into the culture medium during the fermentation process.

Adenosine Triphosphate↗

PubMed1980

Occurrence of a large molecular size form of polyphosphate-glucose phosphotransferase in extracts of Mycobacterium tuberculosis H37Ra.

Extracts prepared by sonicating Mycobacterium tuberculosis H37Ra cells with subsequent centrifugation at 18,000 X g proved to contain a very large molecular size form of polyphosphate-glucose phosphotransferase. The enzyme was separable by polyacrylamide gel electrophoresis, DEAE-cellulose chromatography or ultracentrifugation. When rechromatographed at alkaline pH values, it gave rise to one of the soluble forms of lower molecular weight. The conversion also took place as a result of n-butanol extraction or salting out with ammonium sulfate and heating of dissolved pellet. Under certain conditions the lower-molecular weight enzyme converted to the higher-molecular weight form by association with a hitherto undefined cell constituent. It is assumed that both ionic and hydrophobic forces play a role in this interconversion phenomenon.

Cell Extracts↗

PubMed1980

[Properties of aminoglycoside-3'-phosphotransferases determined by kanamycin transposones Tn 601 and Tn 5].

Aminoglycoside-3'-phosphotransferase I and II (APT-3'-I and APT-3'-II) has been purified to homogenity from the cells of E. coli containing the plasmids R6 and JR67, respectively. The purification procedure involved competitive affinity chromatography on neomycin-sepharose and gel-filtration on Sephadex G-100. The specific activity of APT-3'-I with the substrates--lividomycin A, neomycin B, paromycin, ribostamycin, kanamycins A and B--are 4.3, 2.8, 2.1, 1.6, 0.9 and 0.8 mol/min. mg protein, respectively. The specific activity of APT-3'-II with the substrates--ribostamycin, paromycin, kanamycins A and B, neomycin B--are 8.0, 7.2, 4.0, 4.5 and 3.6, respectively. Mg2+ is required for the activity of both enzymes. Co2+, Zn2+ and Mn2+ are active in case of APT-3'-I; however, these cations are less active than Mg2+. The pH-optimum of APT-3'-I and APT-3'-II is 7.0--7.5. High ionic strength is required for the activity of both enzymes. The molecular weights of APT-3'-I and APT-3'-II are about 36 000 and 26 000, respectively. The amino acid composition of APT-3'-I and APT-3'-II was determined. Both enzymes contain tryptophane residues whose fluorescence intensity decreased when ATP, but not amino-glycoside antibiotics, is added. The interrelationship between the molecular weights of these enzymes and the sizes of the loops of transposones Tn 601 and Tn 5, encoding APT-3'-I and APT-3'-II, is discussed.

Amino Acids↗

PubMed1982

Nucleoside phosphotransferase from barley. Characterization and evidence for ping pong kinetics involving phosphoryl enzyme.

A nucleoside phosphotransferase has been purified to homogeneity from barley seedlings. Its Mr is about 50,000 and it consists of two subunits of equal size. A tightly bound metal ion required for activity can be replaced by Mg, Ba, or Co ions, but not by divalent Cu, Ca, Cd, or Hg. The enzyme is capable of catalyzing the transfer of phosphate from nucleoside monophosphates to the 5'-hydroxyl of any other nucleoside, but shows a decided preference for purine deoxynucleoside phosphate acceptors. A short lived phosphoryl enzyme intermediate has been trapped by rapid denaturation in presence of [32P]AMP. The steady state kinetics of this enzyme is found to be fully consistent with a branched ping pong pathway involving a compulsory phosphoryl enzyme intermediate from which phosphate may be transferred to nucleoside or to water. With appropriately chosen substrates, parallel line reciprocal plots were obtained, provided that both products of the branched pathway were taken into account in the rate measurements. Conversely, when only transphosphorylation was measured, converging reciprocal plots were obtained. Accordingly, assays were devised for the measurement of three velocity components: transphosphorylation, hydrolysis, and the sum of both reactions. Competitive inhibition of the transferase reaction by higher concentrations of either substrate is attributed to deadend interactions. Instead of being inhibited by the formation of a putative E-P.AMP complex, however, the phosphatase reaction is accelerated almost 2-fold.

Adenosine Monophosphate↗

PubMedDate not supplied

[Thymidine kinase and nucleoside phosphotransferase in blood of animals after partial hepatectomy and in patients with inflammatory diseases].

Distinct activation of thymidine kinase (exceeding 2-3-fold the control values) as well as of nucleoside phosphotransferase (about 50% higher as compared with controls) was observed in blood serum of rats within 24 hrs after partial hepatectomy. Within 48 hrs after beginning of the tissue regeneration the activities of both these enzymes were decreased 1.5-fold; the values normalized within 72 hrs. These enzymatic activities were detected in blood of the patients with impairments of liver, kidney and some other tissues.

Animals↗

PubMed1983

Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene.

The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.

Amino Acids↗

PubMed1984

Analysis of the catalytic domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins.

Proteolytic digestion of the transforming protein of Rous sarcoma virus (pp60src) with trypsin, chymotrypsin, or thermolysin generated a 29,000-dalton fragment representing the carboxyl half of this molecule. This proteolytic fragment was able to phosphorylate pp60src-specific immunoglobulin as well as exogenous substrates such as angiotensin, casein, and tubulin. When quantitated on a molar basis, the protease-resistant fragment of pp60src had a greater specific activity than the intact enzyme. Digestion of pp90yes, the transforming protein of Y73 sarcoma virus with these proteases yielded a peptide of similar molecular weight which was capable of autophosphorylation as well as the phosphorylation of exogenous substrates. The proteolytic fragments of both pp60src and pp90yes displayed the same strict specificity for phosphorylation of tyrosine as the intact enzymes. These results indicate that the 29,000-dalton carboxyl end of pp60src and pp90yes can function independently as phosphotransferases and indicate that the catalytic domains of these molecules have a conformation which confers protection against limited conditions of proteolysis.

Animals↗

PubMed1983

The intramembrane topography of the mannitol-specific enzyme II of the Escherichia coli phosphotransferase system.

The D-mannitol-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is an integral cytoplasmic membrane protein responsible for concomitant transport and phosphorylation of this hexitol. We have investigated the intramembrane topography of this enzyme/permease using proteases, membrane-impermeable reagents, and antibodies against the purified protein. The results of these experiments suggest that this protein spans the membrane in a single orientation with a sizeable proportion of its mass extending into the cytoplasm, but with little of the polypeptide exposed at the outside surface of the membrane. Such an orientation is consistent with the reception and transport roles of the mannitol Enzyme II in E. coli.

Cell Membrane↗

PubMed1983

Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease.

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.

Cross Reactions↗

PubMed1984

The primary structure of Salmonella typhimurium HPr, a phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system. A correction.

The protein HPr is a low-molecular-weight phosphocarrier protein of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. We have recently reported the complete primary amino acid sequence of HPr isolated from Salmonella typhimurium (Weigel, N., Powers, D.A., and Roseman, S. (1982) J. Biol. Chem. 257, 14499-14509). This sequence is incorrect at certain residues; the correct primary structure of the protein is presented in this report. The corrected structure generally agrees with the primary sequence predicted for HPr from Escherichia coli (based on the nucleotide sequence of the corresponding ptsH gene). The one apparent ambiguity is at the carboxyl terminus.

Amino Acid Sequence↗

PubMed1982

Bacterial phosphotransferase system. Solubilization and purification of the glucose-specific enzyme II from membranes of Salmonella typhimurium.

The glucose-specific enzyme II (IIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium has been purified to homogeneity. Purification included the following steps: detergent solubilization of membranes in polydisperse octyloligooxyethylene, isoelectrofocusing, chromatofocusing, and either glycerol gradient centrifugation or gel filtration, all in the presence of the same detergent. Enzymatic activity was assayed as phosphoenolpyruvate-dependent phosphorylation of methyl-alpha-D-glucopyranoside. It could be measured after detergent dilution only and required the presence of phosphatidylglycerol in a sonicated suspension. An antiserum prepared against enzyme IIGlc specifically inhibited phosphorylation of methyl-alpha-D-glucopyranoside. In the solubilized state, purified enzyme IIGlc exists as a complex of molecular weight of 105,000 and a sedimentation coefficient of 3.8 S. In polyacrylamide gels in sodium dodecyl sulfate, it has an apparent molecular weight of about 40,000.

Amino Acids↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Primary structure and active site of a general phosphocarrier protein (HPr) from Salmonella typhimurium.

The general histidine-containing phosphocarrier protein (HPr) of the Salmonella phosphotransferase system is required for the phosphorylation of all sugar substrates by this system. The complete amino acid sequence of HPr, consisting of 84 amino acid residues, has been established. The sequence was determined by cleaving the protein with cyanogen bromide, trypsin, and with a protease from Staphylococcus aureus, followed by isolation and amino acid sequence determination of the resulting peptides. The Salmonella typhimurium protein contains two histidine residues, at positions 15 and 75, respectively. The phosphoryl group in phospho-HPr was linked to the His-15 residue. Based on several lines of evidence, the HPr protein from Escherichia coli appears to be identical with the protein from S. typhimurium. The HPr protein from S. aureus has also been isolated in this laboratory and was shown to differ from the HPr proteins described above both with respect to amino acid composition and the inability of the S. aureus and E. coli HPR proteins to substitute for each other in the in vitro sugar phosphorylation assays. The complete amino acid sequence of S. aureus HPr has been reported (Beyreuther, K., Raufuss, H., Schrecker, O., and Hengstenberg, W. (1977) Eur. J. Biochem. 75, 275-286), and its secondary structure has been predicted; this protein contains 70 amino acid residues and only one histidine. In the present studies, three methods were used to predict the secondary structure of S. typhimurium HPr, the results were combined, and a secondary structure for the protein is proposed. Although the amino acid compositions and sequences of the S. typhimurium and S. aureus HPr proteins are quite different, 13 residues are identical in the sequence of the two proteins, and most of these are located near the active site histidine residue. In addition, the predicted secondary structures of the two proteins are quite similar; the additional 14 residues in S. typhimurium, located at the carboxyl terminal end, are predicted to form an alpha-helix.

Amino Acid Sequence↗