Phosphate Transfer Reactions Research Articles

Receptor-stimulated guanine-nucleotide-triphosphate binding to guanine-nucleotide-binding regulatory proteins. Nucleotide exchange and beta-subunit-mediated phosphotransfer reactions.

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[gamma-thio]triphosphate, GTP[S], and guanosine 5'-[beta, gamma-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein beta subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formyl-peptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine-nucleotide-binding regulatory proteins and suggest that, in addition to the nucleotide-exchange reaction, a (thio)phosphate-group-transfer process via guanine-nucleotide-binding regulatory protein beta subunits is involved in the receptor-stimulated binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins.

The family of organo-phosphate transport proteins includes a transmembrane regulatory protein

This review article briefly summarizes aspects of our current understanding of the Uhp sugar phosphate transport system in enteric bacteria, particularly the mode of genetic regulation of its synthesis. This regulation occurs by a process that involves an example of the very widespread and ever-growing group of so-called two-component bacterial regulatory systems, a mechanism of response to environmental signals that employs phosphate transfer reactions between constituent proteins. Of emphasis here is the unusual involvement in transmembrane signaling of the UhpC protein which is related in sequence and structure to some transport proteins, including the very protein whose synthesis it helps regulate.

The anatomy of a kinase and the control of phosphate transfer.

Table of contents Basic features of phosphoglycerate kinase (PGK), The sequences and crystal structures of PGK NMR studies of the structure of the protein PGK, Thermal analysis of yeast PGK, Additional data from NMR studies Substrate binding, Nucleotide binding site: MgATP and MgADP, The function of magnesium, The triose substrate binding site, NMR analysis of substrate binding: summary, General anion‐binding site The conformation change, Kinetic studies of the reaction, Site‐specific mutagenesis studies Effects of mutations on protein structure in the absence of substrates, Mutations in the interdo‐main region, Mutations in the basic patch Effect of Gri3P binding on mutant proteins, Mutations in the interdomain region, Mutations in the basic patch Effects of mutations on kinetics of PGK, Mutations in the interdomain region, Mutations in the basic patchConclusions from experiments with mutants Kinetic effects of low sulphate concentration, Interdomain region, Basic patch region The so‐called hinge of proteins, Features of PGK Common features with other NTP‐utilising systems, The nucleotide‐binding site, The catalytic loop, Domain closure Comments on mechanical devices in multi‐protein enzymes

Prokaryotic elongation factor Tu is phosphorylated in vivo.

Covalent modification of proteins by phosphate transfer reactions constitutes a major mechanism of regulation in higher eukaryotes. Recently, phosphorylation of eukaryotic elongation factors has been described. Analysis of Escherichia coli proteins revealed several of them to be phosphorylated. Various lines of evidence lead us to conclude that one of these proteins is identical to elongation factor (EF) Tu, which can be phosphorylated in vivo at one of its threonine residues. Structural analysis showed that one fragment of the phosphorylated EF-Tu is highly resistant to tryptic digestion. Phosphorylation of eubacterial EF-Tu is not restricted to the E. coli factor but could also be demonstrated for Thermus thermophilus HB8 EF-Tu. Overexpression of tufA did not increase the number of EF-Tu molecules to be phosphorylated. This may indicate that a constant but limited amount of EF-Tu is modified, possibly for a specific function. Phosphorylation of EF-Tu could also be demonstrated in vitro. Upon analysis of subcellular fractions the highest kinase activity was found in the ribosomal fraction of E. coli. Protein sequencing of both the in vivo and in vitro phosphorylated protein revealed position 382 as the modified threonine residue.

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water ( k 3) or a nucleophile ( k 4) is rate-determining. According to this model, in the presence of diethanolamine, k 3 and k 4 were determined to be 4.44 s −1 M −1 and 1000 s −1 M −1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.

Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay

Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress. Activation of sporulation-specific gene transcription is under the control of the spoOA gene product. The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges. Response regulators are activated by specific kinases which phosphorylate them. In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus. The phosphodonor in this reaction is the phosphorylated form of SpoOF. It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress. The series of phosphate transfer reactions in this system is called a phosphorelay. The end product of this series of reactions is SpoOA ∼ P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form. SpoOA ∼ P, but not SpoOA, was shown to be an activator of transcription of the SpoIIA operon which codes for the sporulation-specific sigma factor σ F. Thus, the initiation of sporulation is dependent on SpoOA ∼ P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g. abrB, and activate others, e.g. spoIIA.

The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells.

Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions (Simons, T. J. B. (1979) Nature 281, 337-338). Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C (Weigel, P. H., and Oka, J. A. (1983) J. Biol. Chem. 258, 5089-5094); vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (P i), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC → γATP (0.2 mM/s), P i → γATP (0.2 mM/s) and the conversion βATP → βADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 μmol/min per mg protein) and adenylate kinase (approx. 0.4 μmol/min per mg protein) suggest that the βATP → βADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from P i and the rate of consumption of oxygen atoms (4 · 10 −3 mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.

Determination of equilibrium 18O isotope effects on the deprotonation of phosphate and phosphate esters and the anomeric effect on deprotonation of glucose-6-phosphate

Currently, the authors are investigating the mechanism(s) of phosphate-transfer reaction. In conjunction with these studies, they have determined equilibrium /sup 18/O isotope effects on the deprotonation of phosphate and phosphate esters.

Arsenate induces stress proteins in cultured rat myoblasts.

The induction of stress proteins was examined in rat myoblast cultures by two-dimensional gel electrophoresis. Data obtained by this analysis led to the following observations. (a) Arsenate, which behaves as a phosphate analogue in cellular phosphate-transfer reactions, stresses cultured rat cells and induces the synthesis of a unique set of proteins. (b) Most of the proteins synthesized after the addition of arsenate are identical to proteins synthesized in rat myoblasts in response to heat shock or arsenite stress. (c) However, both arsenic salts induce the synthesis of two unique proteins not induced by heat shock. (d) Five 25-30-kdalton stress proteins of rat cells do not contain methionine residues. (e) A majority of the proteins synthesized in stressed myogenic cells are also induced by stress in other rat cells such as hepatoma cells, pituitary tumor cells, and fibroblasts. The 25-30-kdalton stress-related proteins identified in myogenic cells, on the other hand, are induced in fibroblasts but not hepatoma or pituitary cells.

Purification of a specific reversible tyrosine-O-phosphate phosphatase.

A phosphatase specific for tyrosine-O-phosphate (Tyr-P) was separated from several nonspecific phosphatases present in the third instar larvae of Drosophila melanogaster. The enzyme hydrolyzed L-Tyr-P, with an apparent Km of 0.14 mM, but not D-Tyr-P after being freed from hydrolytic activity toward p-nitrophenyl phosphate, the common phosphatase substrate. Such purified preparations also catalyzed a reversible phosphate transfer reaction from unlabeled Tyr-P to [3H]tyrosine. The transfer activity was L4-14% of the hydrolytic activity, depending on the initial concentration of tyrosine (0.25-4.0 mM). The two activities coincided throughout purification. However, they differed in pH optimum, that of hydrolysis being 6.5-7 and that of phosphate transfer being 7.7.5. The two activities were also differentially inhibited by 1-p-bromotetramisole oxalate in the presence of EDTA and by Mn2+. Addition of Mg2+ did not affect either hydrolysis or phosphate transfer, but 5 mM Zn2+ was 65% inhibitory to both. Sodium fluoride strongly inhibited both reactions, and this inhibition was reversed by EDTA, while EDTA itself had no effect. Pi had no effect and no detectable incorporation of 32Pi into Tyr-P was observed, indicating that the phosphate transfer reaction is not a simple reversal of hydrolysis. No ATP-linked phosphorylation of tyrosine was found.

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.

The three-phase test: intermediates in phosphate transfer reactions

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe three-phase test: intermediates in phosphate transfer reactionsJulius Rebek Jr., Francisco Gavina, and Carmen NavarroCite this: J. Am. Chem. Soc. 1978, 100, 26, 8113–8117Publication Date (Print):December 1, 1978Publication History Published online1 May 2002Published inissue 1 December 1978https://doi.org/10.1021/ja00494a015RIGHTS & PERMISSIONSArticle Views127Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (593 KB) Get e-Alerts Get e-Alerts

The role of sodium ions in ATP formation by the sodium pump

A study has been made to characterize ATP synthesis from ADP and P 1 by the sodium pump with membranes isolated from rabbit kidney. Incorporation of 32 P 1 into ATP was measured. ATP was not formed in a one-step reaction. Synthesis was dependent upon two separate incuba­tions in which incorporation of P 1 into phosphoprotein was followed by transfer of phosphate to ADP. The experimental conditions for the first reaction were different from those for the second (phosphate transfer) reaction. The latter took place only when a high Na concentration (at least 0.5 M) was added. ATP formation was prevented by both K and ouabain. The sequence of reactions could be repeated after washing to remove Na ions, indicating that the driving force for the reactions could be regenerated. The dissipation of ionic gradients was not involved because the Lubrol-solubilized pump was also able to synthesize ATP. It was concluded that the energy for ATP synthesis was derived from specific and reversible interactions of Na ions with the pump. The reactions for ATP synthesis by the sodium pump have been summarized in the form of a cycle.

The state of ADP or ATP fixed to the mitochondria by bongkrekate.

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect. (a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephosphorylation similar as endogenous nucleotides. It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions. The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.

Acid phosphomonoesterase of human prostate. Phosphate transfer reactions and comparison with alkaline phosphatase

The acid phosphomonoesterase I (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) from the human prostate gland was isolated in pure form by an improved procedure. Reaction with [ 32P]orthophosphate at pH values from 3 to 9, followed by protein precipitation with HClO 4, led to no incorporation of phosphate into this enzyme, nor did a substrate affect this. No inhibition by beryllium ion of the enzymic activity occurred, at pH 5 or 7. These two effects are in clear contrast to the behavior of alkaline phosphatase. The competitive transfer of phosphate from p- nitrophenyl phosphate to water or to ethanolamine, catalyzed by phosphatase, was measured. With Escherichia coli alkaline phosphatase, the phosphate transferred exchanged to a significant extent with external [ 32P]orthophosphate, at pH 9. With the acid phosphatase no such exchange occurred, nor did that enzyme catalyze an exchange of orthophosphate with O- phosporylethanolamine when hydrolyzing the latter. the evidence indicates that if a phosphoyl-enzyme intermediate is involved at all in prostatic acid phosphatase catalysis, it is to quite a different type to that in the known alkaline phosphatases.

The binding of the calcium transport inhibitors reserpine, chlorpromazine and prenylamine to the lipids of the membranes of the sarcoplasmic reticulum.

1. Reserpine, chlorpromazine, prenylamine and imipramine are bound to vesicles isolated from the sarcoplasmic reticulum of rabbit striated muscle cells as well as by the lipids which can be extracted from the vesicular preparations. 2. At concentrations of 5×10−7 M reserpine, chlorpromazine and prenylamine, drug binding can just be detected. At drug concentrations of 10−4 M the drug uptake amounts to 0.1 μmol per mg vesicular protein, 0.2 μmol drug per mg vesicular lipid and ∼0.4 μmol drug per mg phosphatidylcholine. 3. Drug binding is reversible and temperature independent. 4. Drug binding is not diminished when the phospholipids are hydrolysed by phospholipase A, C or D. 5. Drug binding is abolished by hydrogenation of the isolated lipids as well as of the lipids bound to the membranes. 6. In addition calcium uptake the Ca++ induced increase in ATP breakdown and the phosphate transfer reaction are inhibited when the lipids in the membranes are hydrogenated. 7. The normal ATPase as well as the lipid free proteins myokinase and actin are not affected by hydrogenation.

Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: XIV. INTERACTION OF PURIFIED MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE FROM BAKERS' YEAST WITH SUBMITOCHONDRIAL PARTICLES FROM BEEF HEART

Abstract 1. A Mg++-dependent adenosine triphosphatase was solubilized and purified from bakers' yeast mitochondria. The enzyme resembled mitochondrial ATPase from beef heart with respect to substrate specificity, cold lability, and other physical properties. 2. An antiserum against the purified yeast enzyme inhibited the ATPase activity of the soluble enzyme as well as ATPase and oxidative phosphorylation in submitochondrial yeast particles. Mitochondrial ATPase from beef heart or from Neurospora crassa was not inhibited by the antiserum. 3. Submitochondrial beef heart particles devoid of endogenous ATPase could bind the purified yeast enzyme without changing its immunological specificity. The ATPase activity of the resultant hybrid particles, like that of beef heart particles, was strongly inhibited by low levels of rutamycin. In contrast, submitochondrial particles from yeast were much less sensitive to this inhibitor. 4. The yeast enzyme stimulated oxidative phosphorylation in beef heart particles which were deficient in, but not devoid of, endogenous ATPase. The stimulation was dependent on the presence of beef heart coupling factor 1 (F1) in these particles and was unaffected by the antiserum against the yeast enzyme. Antiserum against beef heart F1 strongly inhibited phosphorylation. These results suggest that yeast F1, in contrast to beef heart F1, does not significantly participate in phosphate transfer reactions when it functions as a coupling factor in beef heart particles. Rather, it is proposed that the stimulation by yeast F1 is due to an effect on the membrane structure.

Bacterial Synthesis of Nucleotides

General properties of bacterial nucleoside phosphotransferase were demonstrated. Nucleoside phosphotransferase activity was observed somewhere in cells, and the activity and the specificity for donor and product in this reaction are described to be due to the basic character of strains. Such aromatic phosphates as p-nitrophenylphosphate, phenylphosphate, benzylphosphate and the nucleotides were apparent to be useful for nucleotide synthesis, and the ability as donor did not always depend upon the energy consideration. The product specificity of this reaction was confirmed to correlate with nucleotide isomer added as donor; that is, the bacteria characterized to phosphorylate at 5′-position of nucleoside catalyzed the interconversion of phosphoryl or phosphate radical between 5′-nuclotides and those characterized to do at 3′(& 2′)-position of nucleoside catalyzed the interconversion of that between 3′(& 2′)-nucleotides. The phosphoryl or phosphate transfer reaction using nucleotide as donor is reversible b...

Phosphate transfer from adenosine diphosphate phosphoglycerate and 2,3-diphosphoglycerate

The following phosphate transfer reaction has been found to be catalyzed by muscle and yeast extracts: