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PubMed1984

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine.

Nucleoside phosphotransferase (nucleotide: 3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a component with a marked fall in catalytic activity. The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and protecting effect of nucleotides, particularly the diphosphate forms. The dissociated form displays lower Vmax and higher S0.5 than the associated ones; and the Hill constants are always about 1. With this form, nucleotides show only a modest activating effect and do not protect. Mg2+, Mn2+ or Co2+ are required for catalytic activity, whereas the protective effect of nucleotides is independent of divalent metals. Inorganic phosphate stabilizes associated forms of the enzyme, but inhibits its activity by competing with nucleotide effectors. The enzyme behaves also as a phosphohydrolase, particularly with respect to deoxyribonucleoside monophosphates; deoxyuridine and deoxythymidine inhibit hydrolytic activity.

Alcohols↗

PubMed1976

A Limulus glucose-6-phosphatase with phosphotransferase activity characteristic of vertebrate liver microsomes. Its possible evolutionary significance.

1. Limulus hepatopancreas, coxal glands and intestine contain a particulate enzyme which can synthesize glucose 6-phosphate from glucose and inorganic pyrophosphate or carbamyl phosphate as well as hydrolyze glucose 6-phosphate. This has been clearly differentiated from hydrolysis by lysosomal or soluble phosphatases. 2. The enzyme resembles vertebrate glucose-6-phosphatase in its specific anatomical distribution, pH optimum, kinetic properties, donor specificity and phospholipid dependence, as indicated by its satency and lability to detergent treatment. 3. A variety of other invertebrates tested exhibited little or no PPi-glucose phosphotransferase activity with these properties. A similar phosphotransferase activity of lobster hepatopancreas had somewhat different kinetic properties and pH optimum. 4. The hypothesis that a specific glucose-6-phosphatase is to be found only in those animals which utilize free glucose as an important circulating form of energy is presented and discussed. It appears that a variety of transport compounds, such as trehalose and glucose, was tried at the evolutionary level of the Arthropods.

Animals↗

PubMed1982

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5.

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3'-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated Mr of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).

Amino Acids↗

PubMed1997

Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides.

Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3'-azido-3'-deoxy-thymidine (AZT), cytosine-beta-D-arabinofuranoside (AraC) and 5-fluoro-2'-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxynucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.

Erythrocytes↗

PubMed1986

Phospholipid transmethylation and choline phosphotransferase in microsomal fraction of human diseased liver.

The activities of three enzymes catalyzing the production or degradation of phosphatidylcholine, a major structural phospholipid of cell membranes, were assessed in hepatocyte membrane microsomal preparation from patients with various types of liver disease. Choline phosphotransferase activity of preparation from patients with chronic aggressive, chronic active, chronic persistent, alcoholic hepatitis and cirrhosis accompanied by marked necrosis and relatively slight fibrosis was markedly decreased, compared with normal liver; the activity from patients with fatty liver and chronic inactive hepatitis was slightly decreased. Specimens from patients with acute transient hepatitis were not significantly different from normal. Methyltransferase and phospholipase A2 activities tended to parallel that of choline phosphotransferase, although the degree of changes was generally less marked. Our studies indicate that enzyme activities that are critical for hepatic cell membrane integrity and activity are attenuated in liver specimens from patients with disease in which there is marked hepatic cell necrosis.

Adult↗

PubMed1995

Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia.

Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.

Adenosine Diphosphate↗

PubMed1985

Bacterial phosphotransferase system: regulation of mannitol enzyme II activity by sulfhydryl oxidation.

The mechanism by which the oxidation-reduction potential regulates the bacterial phosphotransferase system in Escherichia coli has been investigated. Transphosphorylation experiments verified that the oxidizing agent, potassium ferricyanide, directly inhibits mannitol enzyme II activity. Phosphorylation of enzyme IImtl with enzyme I, heat-stable phosphocarrier protein of the phosphotransferase system, and phosphoenolpyruvate partially protects the enzyme from ferricyanide inhibition. The enzyme is even less sensitive to inhibition during catalytic turnover. Preincubation of unphosphorylated enzyme with ferricyanide, however, reversibly inactivates it even at high mannitol concentrations. The results are inconsistent with a regulatory mechanism in which sulfhydryl oxidation influences the affinity of the enzyme for the substrate. Instead, it is concluded that the oxidized enzyme is inactive.

Escherichia coli↗

PubMed1985

Bacterial phosphotransferase system: regulation of the glucose and mannose enzymes II by sulfhydryl oxidation.

We have investigated the effect of oxidizing agents on methyl alpha-glucoside phosphorylation by the Escherichia coli phosphotransferase system (PTS). Oxidizing agents inhibited methyl alpha-glucoside phosphorylation at low methyl alpha-glucoside concentrations, and the degree of inhibition was shown to decrease with increasing concentrations of methyl alpha-glucoside. Results of studies with mutant bacteria and substrate analogues of the glucose and mannose enzymes II showed that contrary to the interpretation of Robillard and Konings [Robillard, G. T., & Konings, W. N. (1981) Biochemistry 20, 5025-5032] the apparent change in the Km value for methyl alpha-glucoside phosphorylation induced by sulfhydryl oxidation is not due to the formation of a low-affinity, oxidized form of the glucose enzyme II. Rather, the results are explained by the presence of two phosphotransferase systems that phosphorylate methyl alpha-glucoside with different affinities and that are differentially sensitive to oxidizing agents. The low Km system corresponds to the glucose enzyme II, which is strongly inhibited by potassium ferricyanide, phenazine methosulfate, and plumbagin. The high Km system corresponds to the mannose enzyme II, which is less sensitive to inhibition by these oxidizing agents. This differential sensitivity to inhibition by oxidizing agents can account for the apparent Km change for methyl alpha-glucoside phosphorylation reported by Robillard and Konings. The physiological significance of sulfhydryl oxidation in the enzymes II of the PTS has yet to be ascertained.

Deoxyglucose↗

PubMed1988

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.

Amino Acid Sequence↗

PubMed1990

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study.

The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

Bacillus cereus↗

PubMed1981

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme i phosphorylation.

The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H--2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C--12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect (nu V/nu D = 1.9), showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse second-order rate constants of the reaction were determined to be 3 X 10(7) and 8 X 10(5) M-1 min-1, respectively, yielding an equilibrium constant of approximately 40 and a delta G degree for enzyme I phosphorylation of --2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.

Binding Sites↗

PubMed1997

Protein phosphorylation chain of a Bacillus subtilis fructose-specific phosphotransferase system and its participation in regulation of the expression of the lev operon.

The proteins encoded by the fructose-inducible lev operon of Bacillus subtilis are components of a phosphotransferase system. They transport fructose by a mechanism which couples sugar uptake and phosphoenolpyruvate-dependent sugar phosphorylation. The complex transport system consists of two integral membrane proteins (LevF and LevG) and two soluble, hydrophilic proteins (LevD and LevE). The two soluble proteins from together with the general proteins of the phosphotransferase system, enzyme I and HPr, a protein phosphorylation chain which serves to phosphorylate fructose transported by LevF and LevG. We have synthesized modified LevD and LevE by fusing a His-tag to the N-terminus of each protein allowing rapid and efficient purification of the proteins. We determined His-9 in LevD and His-15 in LevE as the sites of PEP-dependent phosphorylation by isolating single, labeled peptides derived from 32P-labeled LevD, LevD(His)6, and LevE(His)6. The labeled peptides were subsequently analyzed by amino acid sequencing and mass spectroscopy. Mutations replacing the phosphorylatable histidyl residue in LevD with an alanyl residue and in LevE with a glutamate or aspartate were introduced in the levD and levE genes. These mutations caused strongly reduced fructose uptake via the lev-PTS. The mutant proteins were synthesized with a N-terminal His-tag and purified. Mutant LevD(His)6 was very slowly phosphorylated, whereas mutant LevE(His)6 was not phosphorylated at all. The corresponding levD and levE alleles were incorporated into the chromosome of a B. subtilis strain expressing the lacZ gene under control of the lev promoter. The mutations affecting the site of phosphorylation in either LevD or LevE were found to cause constitutive expression from the lev promoter of B. subtilis.

Bacillus subtilis↗

PubMed1994

Four monoclonal antibodies inhibit the recognition of arylsulphatase A by the lysosomal enzyme phosphotransferase.

The critical step in the sorting of lysosomal enzymes is their recognition by a phosphotransferase in the Golgi apparatus. The topogenic sequences responsible for the recognition by this enzyme have so far only been defined for the lysosomal protease cathepsin D. We have generated four monoclonal antibodies directed against lysosomal arylsulphatase A (ASA). These antibodies inhibit the recognition of ASA by the phosphotransferase in vitro and thus define a region of topogenic sequences in the ASA polypeptide. The antibodies do not interfere with the enzymic activity nor with pH-dependent dimerization of ASA. The epitopes recognized by the antibodies have been located in the second quarter of the ASA polypeptide using chimeric mouse-human ASA molecules. Three of the monoclonal antibodies bind to identical or closely adjacent epitopes, which are formed by the interaction of amino acid residues 165-184 and 202-240. The fourth antibody recognizes a different epitope within amino acids 256-265.

Amino Acid Sequence↗

PubMed1997

Phage display selection of peptides against enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system (PTS).

The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. Uniqueness and pleiotropic function make the PTS a target for new antibacterial drugs. Enzyme I is the first component of the divergent protein phosphorylation cascade of the PTS. It transfers phosphoryl groups from phosphoenolpyruvate to the general phosphoryl carrier protein HPr. Six 15-mer, nine 10-mer and nine 6-mer peptides that inhibit enzyme I were selected from phage display libraries. Of these, 16 were synthesized and characterized. The majority of the peptides contain a histidine with an adjacent arginine. Two peptides were found to contain cysteines but no histidine. All peptides are rich in basic residues and lack acidic amino acids. The peptides inhibit the phosphotransferase system in vitro with IC50 of between 10 microM and 2 mM. Some, but not all, of the peptides inhibit cell growth in the agar diffusion test by an as yet undefined mechanism. All peptides are phosphorylated by enzyme I, and some are regenerated by slow autocatalytic hydrolysis of the phospho-peptide bond.

Amino Acid Sequence↗

PubMed2001

Precise amounts of a novel member of a phosphotransferase superfamily are essential for growth and normal morphology in Caulobacter crescentus.

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10-fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P-type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site-directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.

Adenosine Triphosphatases↗

PubMed1969

Regulatory effects of substrates on a phosphotransferase from Aerobacter aerogenes and modification of its activity by partial proteolysis.

An enzyme isolated from Aerobacter aerogenes acts both as phosphomonoesterase and phosphotransferase with glucose, glucosides, glucosamine, and N-acetyl glucosamine as acceptors. When glucose-6-phosphate is the phosphate donor, these acceptors appear to act as activators of the enzyme, while with p-nitrophenyl phosphate, alpha-glycerophosphate, fructose-1,6-diphosphate, and several other phosphate esters as donors, the same acceptors act as noncompetitive inhibitors. With p-nitrophenyl phosphate as the phosphate donor, no inhibition is observed when glucose is replaced as acceptor by 2-deoxy glucose. The inhibition by glucose and other acceptors is eliminated at low pH or by increasing the temperature of reaction. Partial proteolysis of the enzyme by chymotrypsin produces a modified enzyme (gl-phosphotransferase-Ch) that shows altered relative velocities for the hydrolysis of several substrates as well as altered regulatory effects by acceptors.

Chymotrypsin↗

PubMed1982

Regulation of lactose permease activity by the phosphoenolpyruvate:sugar phosphotransferase system: evidence for direct binding of the glucose-specific enzyme III to the lactose permease.

Interaction between the glucose-specific enzyme III (enzyme IIIglc) of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose permease was studied with membrane fragments from an Escherichia coli strain that overproduces the lactose permease. Substrates of the permease markedly and specifically stimulated binding of enzyme IIIglc to the membranes. The sugar-stimulated binding of enzyme IIIglc was concluded to the specific to the lactose permease because it (i) was dependent on the amount of the permease, (ii) was promoted only by sugar substrates of the permease, and (iii) was completely eliminated by treatment of the membranes with N-ethylmaleimide in the absence (but not the presence) of thio-beta-D-digalactoside. The pH dependence of binding was similar to that reported for the binding of thio-beta-D-digalactoside to the permease. Phosphoenolpyruvate prevented the binding of enzyme IIIglc to the lactose permease in the presence (but not the absence) of the other phosphate transfer components of the phosphotransferase system. These results support the hypothesis that enzyme IIIglc, in its dephosphorylated form, modulates the activity of the lactose permease by a direct protein-protein interaction.

Allosteric Regulation↗

PubMed1987

Bacillus subtilis sucrose-specific enzyme II of the phosphotransferase system: expression in Escherichia coli and homology to enzymes II from enteric bacteria.

Sucrose is transported into Bacillus subtilis cells by way of a phosphotransferase system, which consists of a specific enzyme II, a nonspecific enzyme I, and a histidine-containing phosphocarrier protein. Mutations in the sacP locus abolish the specific transport of sucrose. The B. subtilis sacP gene was cloned and expressed in Escherichia coli, and transformed cells could transport and phosphorylate sucrose. This indicates that the sacP gene product is enzyme II of the sucrose phosphotransferase system of B. subtilis. The nucleotide sequence of the sacP gene was determined and was found to overlap with the sacA gene at the tetranucleotide ATGA, which may allow a translational coupling between sacP and sacA. The two genes are therefore probably organized in an operon structure with the promoter located 5' to sacP gene. The deduced amino acid sequence gave a Mr of 48,945 for the sucrose-specific enzyme II polypeptide. The amino acid sequence was compared to that of three other known enteric bacterial enzymes II (beta-glucoside-specific enzyme II, mannitol-specific enzyme II, and glucose-specific enzyme II). Homology was found with beta-glucoside enzyme II, and well conserved regions were identified through the comparison of the proteins.

Amino Acid Sequence↗