Transfer Of Phosphate Groups Research Articles

Identification of the gene ( aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product

An open reading frame located in the tyrB- uvrA intergenic region of the Escherichia coli MG1655 chromosome was identified as encoding the class B acid phosphatase of this species on the basis of cloning and expression experiments. A protocol for purification of the enzyme (named AphA) was developed, and its properties were analyzed. The enzyme is a 100-kDa homotetrameric protein which apparently requires a metal co-factor for activity. Similarly to other bacterial class B acid phosphatases, it is able to dephosphorylate several organic phosphomonoesters as well as to catalyze the transfer of low-energy phosphate groups from phosphomonoesters to hydroxyl groups of various organic compounds.

14 IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF HUMAN INTESTINAL CREATINE TRANSPORT ACTIVITY.

Creatine kinase reversibly catalyzes the transfer of high-energy phosphate groups from phosphocreatine to ADP to regenerate ATP in tissues with high and fluctuating energy demands, including a number of polarized cell types. Having previously identified significant creatine kinase activity in human intestinal epithelium and in CACO-2 cells, we were interested in further characterizing creatine transport activity in the human intestine. Utilizing the reported sequence of the rabbit creatine transporter, we cloned and sequenced a human creatine transporter (CTI) from a human intestinal cDNA library. Northern analysis revealed expression in human brain, muscle, and intestine. CT1 was mapped to Xq28 utilizing somatic cell hybrids and FISH. During these experiments a second creatine transporter sequence, not expressed in intestine, was identified and mapped to 16p11.2 utilizing YACS and cosmid contigs from these YACS. CT1 was shown by RT-PCR to be expressed at high levels in human gastric, intestinal, and colonic epithelium and in CACO-2 and T84 cells. In CACO-2 cells, CT1 expression was regulated by available creatine and 14C-creatine uptake studies demonstrated saturable kinetics. Depletion of phosphocreatine in confluent T84 monolayers resulted in significant reductions of short circuit current following stimulation with forskolin alone or forskolin and carbachol. These findings demonstrate a role for creatine transport in intestinal cellular energy metabolism. (Supported by the Nemours Foundation)

Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

The ‘CheA’ and ‘CheY’ domains of Myxococcus xanthus FrzE function independently in vitro as an autokinase and a phosphate acceptor, respectively

FrzE is a chemotaxis protein in Myxococcus xanthus which has sequence homology to two different chemotaxis proteins of enteric bacteria, CheA (autokinase) and CheY (phosphate acceptor) [Proc. Natl. Acad. Sci. USA 87 (1990) 5898–5902]. It was also shown that a recombinant FrzE protein was autophosphorylated when incubated in the presence of ATP and Mn 2+ [J. Bacteriol. 172 (1990) 6661–6668]. In this study, we further investigated the biochemical properties of FrzE. Two recombinant proteins were produced: one containing only the ‘CheA’ domain of FrzE and the second only the ‘CheY’ domain. The CheA domain polypeptide contained the autokinase activity which was absent from the CheY domain polypeptide. The phosphorylated CheA domain polypeptide as well as the intact FrzE protein were able to transfer phosphate groups to the CheY domain peptide. These results indicate that FrzE has structural as well as functional homologies to CheA and CheY in a single polypeptide.

Mechanisms of Magnesium Transport

Mg, the second or third most abundant intracellular cation, is an important constituent of cells and is a necessary cofactor for many enzymes especially those involved in the transfer of phosphate groups. In this role Mg is essential for Na and Ca pump activity. Recently it has been shown to regulate the activity of the Na,K,CI (18) and K,CI (39) co-transport systems and to influence ionic traffic through Ca (34), K (37), and Na (48) channels. Mg may thus be an important link between ion transport and metabolism, and it is likely that its cytoplasmic concentration is precisely regulated. This chapter will focus on how Mg crosses the membrane and on some of the factors that influence the process. It will show how models initially formulated to de­ scribe Mg transport in squid axon and barnacle muscle have been used to explain recent observations on red cells. The earliest experiments on Mg transport suggested it was difficult to change cell Mg content. Several cell types had been incubated for long periods in media containing either high Mg concentrations or Mg chelators without significantly affecting cell Mg content. This, together with the observation that 28Mg equilibrated very slowly across some cell membranes, suggested that membranes had a particularly low Mg permeability (17, 19)

Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer.

Signal transduction in Escherichia coli involves the interaction of transmembrane receptor proteins such as the aspartate receptor, Tar, and the products of four chemotaxis genes, cheA, cheY, cheW, and cheZ. It was previously shown that the cheA gene product is an autophosphorylating protein kinase that transfers phosphate to CheY, whereas the cheZ gene product acts as a specific CheY phosphatase. Here we report that the system can be reconstituted in vitro and receptor function can be coupled to CheY phosphorylation. Coupling requires the presence of the CheW protein, the appropriate form of the receptor, and the CheA and CheY proteins. Under these conditions the accumulation of CheY-phosphate is enhanced approximately 300-fold. This rate enhancement is seen in reactions using wild-type and "tumble" mutant receptors but not "smooth" mutant receptors. The increased accumulation of phosphoprotein was inhibited by micromolar concentrations of aspartate, using wild-type, but not tumble, receptors. These results provide evidence that the signal transduction pathway in bacterial chemotaxis involves receptor-mediated alteration of the levels of phosphorylated proteins. They suggest that CheW acts as the coupling factor between receptor and phosphorylation. The results also support the suggestion that CheY-phosphate is the tumble signal.

Src-related protein tyrosine kinases.

Tyrosine protein kinases (TPKs) catalyze the transfer of phosphate groups from nucleotide triphosphates to tyrosine residues on proteins and peptides. While phosphorylation of proteins on tyrosine residues is a well-known post-radiational event detected in different regulatory systems, only a small number of cellular proteins are known substrates for TPKs [1]. Coupled with the likelihood that many of these modifications are rapidly turned over, it is not surprising that the abundance of phosphotyrosine in most cells is less than 0.05% of the steady-state phosphoamino-acids [2]. There are about 30 mammalian TPKs that have been identified to date (table 1); however, this number is expected to increase as strategies for cloning new genes belonging to this group become more refined. Most TPKs can be divided into two major groups (table 1): those corresponding to growth factor receptors (such as the epidermal growth factor receptor) and those associated with the internal portion of the plasma membrane (such as the c-src protein). The growth factor receptors are covered elsewhere (see chapters 8 and 9).KeywordsRous Sarcoma VirusTyrosine Kinase GeneAvian Sarcoma VirusTransform NIH3T3 FibroblastThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity.

The periplasmic nonspecific acid phosphatase II from Salmonella typhimurium was purified to homogeneity from a mutant strain that overproduces the enzyme (Uerkvitz, W., and Beck, C.F. (1981) J. Biol. Chem. 256, 382-389). It was shown that the enzyme transfers phosphate groups from organic phosphoric acid esters (donors) to water as well as to the 2'-, 3'-, or 5'-hydroxyls of nucleosides, nucleotides, and other compounds with free hydroxyl groups (acceptors). The enzyme was crystallized in two forms by precipitation with polyethylene glycol. Needles were formed in buffer containing Mg2+, whereas thin rectangular plates appeared in the presence of the non-ionic detergent n-octyl glucoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under partially or completely denaturating conditions revealed that the native enzyme is a tetramer consisting of identical 24-kDa monomers. Owing to surface inactivation, polyethylene glycol, non-ionic, or Zwitterionic detergents are indispensable for enzyme stability. The detergents are able to reactivate inactivated enzyme when present near or above their critical micelle concentration.

Phosphorylation and Dephosphorylation Reactions of the Red Beet Plasma Membrane ATPase Studied in the Transient State

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([gamma-(32)P]ATP and [5',8-(3)H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg(2+)-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg(2+) increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.

Fucokinase, its anomeric specificity and mechanism of phosphate group transfer

Fucokinase phosphorylates l-fucose at the anomeric position and, as such, might use either the α or β anomer as its substrate. Examination of the utilization of radiolabelled α and α,β mixtures established β- l-fucose as the required substrate. Phosphorylation at the anomeric center might involve either the loss or retention of the anomeric oxygen. The mechanism has been shown to involve anomeric oxygen retention through mass spectrometric analysis of the product phosphate derived from 18O-labelled l-fucose.

Purification and properties of the ATP: adenylylsulphate 3?-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3′-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg2+ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.

Determination of levels of glycolytic intermediates and nucleotides in platelets by pulse-labeling with [ 32P]orthophosphate

A method is presented for the separation and quantification of 32P-labeled carbohydrates and nucleotides in blood platelets which have been pulse-labeled with [ 32P]orthophosphate. The procedure is based on two-dimensional paper chromatography, identification of the spots by radioautography and enzymatic methods, and quantitation of 32P radioactivity by liquid scintillation counting. The data show that 32P is homogeneously distributed among the compounds studied so that the total radioactivity is proportional to the levels of these compounds in the metabolic compartment of the cells. Thus, this method provides a sensitive and accurate means to evaluate phosphorylated intermediates in glycolysis and nucleotide metabolism and to assess the transfer of energy-rich phosphate groups between these pathways in particular.

X-ray structure of the ternary complex Zn(II)-ATP-2,2'-bipyridyl and possible model for ATP transport.

Ternary complexes between metal ions, nucleotides and aromatic heterocyclic amines are being investigated by X-ray diffraction as possible models for enzyme-metal-substrate interactions. Kennard and coworkers have reported the crystal structure of the ATP disodium salt. Although more recently several crystal structures of binary and ternary complexes between metal ions and nucleoside monophosphates have been reported, no structure combining metal complexes with nucleoside di- or triphosphates has been reported. Most divalent metal ions catalyse the nonenzymatic transfer of phosphate from nucleoside polyphosphates to various acceptors. Therefore, these compounds in water solution undergo extensive hydrolysis in the presence of metal ions, leading to the formation of the monophosphates; for instance, at pH 6.5, Cu2+ accelerates the hydrolysis of ATP by a factor of about 300 (ref. 5). We report here the crystal and molecular structure of the complex [Zn(II)-H2ATP-2,2'-bipyridyl]2.4H2O, determined by single crystal X-ray analysis. The complex molecule provides a possible model for ATP transport and phosphate group transfer mechanism.

Crystal and molecular structure of the ternary complex Zn(II)–ATP–2,2′-bipyridyl

A series of microcrystalline compounds between adenosine 5′-triphosphoric acid (ATP), 22′-bipyridyl and some 3d metal ions, such as Mn(II), Co(II), Cu(II) and Zn(II), have been obtained in 1:1 ratio [1]. Crystal suitable for X-ray analysis were obtained for the zinc compound. Diffractometer data, collected on one of these crystals, gave the following results: a = 11.105(3), b = 25.223(7), c = 1.539(3) Å, β = 91.34(4)°, monoclinic, space group P2 1. 1617 reflections with intensity greater then twice their ▪ standard deviation were used for the structure determination and refinement ( R = 0.098). The structure of the compound consists of dimeric molecules in which two zinc atoms are held together by two –OPO– bridges from the γ-phosphate groups of two ATP molecules (Fig. 1). Both zinc atoms show a distorted octahedral coordination, formed by two oxygen atoms from different γ-phosphate groups, one oxygen atom from the β-phosphate group and the two nitrogen atoms of the bipyridyl ligand. The sixth position is completed by an α-phosphate oxygen atom which is only weakly bound. ZnN and ZnO distances average respectively 2.15(4) and 2.0293) Å. The structure is held together by strong intermolecular bipyridyl–purine and bipyridyl–bipyridyl stacking interactions. Weaker bipyridyl–purine intramolecular stacking is also observed. The molecule provides a possible model for ATP transport and phosphate group transfer mechanism.

Conditions of occurrence for primeval processes of transphosphorylations

Model reactions of the phosphate group transfer through hydrolysis-condensation processes without enzymic catalysts are studied in dilute aqueous solutions. Tripolyphosphate and acetylphosphate are used as energy and phosphate donors to perform syntheses of pyrophosphate. Results are discussed in terms of the variation versus pH of the overall standard free enthalpy change.

Further studies on DNA thermosensitive mutants of Escherichia coli using the toluenized cell system

In vitro DNA synthesis was analyzed in toluenized cells of several DNA thermosensitive mutants, with special emphasis on ATP requirement. The results obtained suggest that ATP acts through the transfer of phosphate groups, and that this transfer must take place during dNTPs incorporation. ATP hydrolysis is independent of the presence of K + . Availability of rNTPs other than ATP does not seem to be a limiting factor for dNTPs incorporation. However, it was found that 3 H-UTP incorporation into a TCA insoluble fraction takes place in the absence of exogenous CTP or GTP, but it requires ATP. This fact suggests a possible role for ATP in DNA replication. In toluenized cells phenotypic reversion of thermosensitivity by salts could not be detected. The behaviour at different temperatures of several DNA thermosensitive mutants containing the polAl mutation was studied, and from the comparison with in vivo properties functions are suggested for the different gene products. La synthèse du DNA in vitro a été analysée dans les cellules de plusieurs mutants DNA thermosensibles, après un traitement au toluène. Une particulière attention a été portée sur les besoins en ATP de ce système. Les résultats obtenus suggèrent que l'ATP agit dans le transfert de groupements phosphate et que ce transfert doit avoir lieu au cours de l'incorporation des dNTPs. L'hydrolyse de l'ATP est indépendante de la présence d'ions K + . La présence de rNTPs autres que l'ATP ne parait pas être nécessaire pour l'incorporation des dNTPs. Cependant nous avons trouvé que l'incorporation de 3 H-UTP dans la fraction insoluble au TCA a lieu en l'absence de CTP ou de GTP exogène mais requiert la présence d'ATP. Ce fait suggère un rôle possible de l'ATP dans la replication du DNA. Dans les cellules traitées au toluène, il n'a pas été possible de détecter de réversion phénotypique de la thermosensibilité par les sels. Le comportement de plusieurs mutants DNA thermosensibles contenant la mutation polAl, a été étudié à différentes températures. La comparaison avec les propriétés in vivo de ces mutants a permis de suggérer des fonctions pour les produits des différents gènes.

Trois classes de systèmes de transport chez les bactéries

The study of sugar transport mechanisms in bacteria failed to reveal an universal principle. In the present diversity, three classes of different mechanisms seem to emerge. The first class of which the lactose permease of E. coli serves as a prototype, uses a unique membrane protein, that behaves like a mobile carrier. The fundamentally symetrical function of such a carrier is made asymetrical by the energy coupling, so that the final result is a unidirectional active transport. It is suggested, that the energy coupling step makes the substrate site of the carrier unavailable for exit. The second class includes vectorial reaction systems, of which the PEP-glucose phosphotransferase system of E. coli is taken as example. Two sequential reactions of phosphate group transfer lend a solid biochemical and thermodynamic background to the understanding of transport, however the nature of the vectorial reaction itself and in particular the structural orientation of enzyme II in the membrane remain obscure. The possibility of a built-in orientation versus an orientation induced by the compartmentation of the soluble components of the system is discussed. The third class of transport systems includes a periplasmic binding protein. The example of galactose binding protein shows, that the association-dissociation kinetics are inadequate to account for the transport properties without the contribution of a postulated membrane protein, the existence of which is also suggested by genetic evidence. L'étude du mécanisme des trnasports actifs de sucres chez les bactéries n'a pas permis de dégager un principe universel. Dans la diversité actuelle, trois classes de mécanismes semblent émerger. La première, dont la lactose perméase d'E. coli est le prototype, fait appel à une protéine membranaire unique se comportant comme un transporteur mobile. Le fonctionnement symétrique d'un tel transporteur est rendu asymétrique par le couplage énergétique aboutissant à un transport unidirectionnel thermodynamiquement actif. Il est suggéré que l'effet du couplage énergétique est de rendre le site d'affinité du transporteur pour son substrat inaccessible pour le transport en retour. La seconde classe comprend les systèmes à réaction vectorielle dont le PEP-glucose phosphotransférase d'E. coli est pris pour l'exemple. Deux réactions de transphosphorylation successives donnent une base biochimique et énergétique solide à la compréhension de ce transport, mais la nature de la réaction vectorielle et en particulier l'orientation structurale dans la membrane de l'enzyme qui en est responsable reste obscure. La possibilité d'une orientation intrinsèque ou d'une orientation imposée par la présence unilatérale des composants solubles du système est discutée. La troisième classe de système de transport fait appel à des protéines périplasmiques porteuses de sites d'affinité pour le substrat transporté. L'exemple du galactose binding protein est utilisé pour montrer que les caractéristiques cinétiques d'association-dissociation de cette protéine sont inadéquates pour expliquer le transport sans le concours d'une protéine membranaire hypothétique suggéré par les études génétiques.

A study of the hormonal control in vitro of Na +-K + activated ATPase of isolated liver plasma membrane

The activity of rat liver plasma membrane-bound Na +-K + ATPase was modulated in vitro by epinephrine, glucagon and insulin through an action probably mediated by cyclic AMP. Epinephrine, glucagon and cyclic AMP inhibited the activity of this enzyme; insulin did not influence the baseline activity but prevented partially the effect of the former hormones. Rat liver plasma membranes contain protein kinase activity. In the absence of exogenous substrate, phosphorylation of membrane constituents occurred very rapidly and was inhibited by cyclic AMP, both in the presence and absence of Ca ++ ions. When an exogenous substrate (protamine) was added to the system, phosphorylation was linear with time, at least up to 15 min; the reaction was stimulated by cyclic AMP, and the effect of this compound was abolished when Ca ++ was added to the medium. Hydroxylamine removed about 35% of labeled phosphate incorporated—both in the presence and absence of cyclic AMP—in membrane preparations incubated in the absence of protamine, while it had no effect in its presence. These results suggest that, as a consequence of endogenous phosphorylation, part of labeled phosphate was incorporated in membranes through an acyl linkage. As Na +-K + ATPase catalyzes two main reaction steps, the first being represented by a phosphate group transfer from ATP to a free carboxylic radical belonging to enzyme protein, these results very probably indicate that the marked effect exerted by cyclic AMP on ATPase activity may be ascribed to the inhibition of the formation of such a linkage, due to the cyclic nucleotide itself. The second step of ATPase reaction was investigated by model reactions. The hydrolysis of p-nitrophenyl phosphate was not modified by cyclic AMP while that of acetyl phosphate was inhibited. As in the latter case, the reaction concerns the hydrolysis of an acyl phosphate group, it seems probable that also the second step of ATPase reaction is inhibited by cyclic AMP.

Phosphate transfer between 2,3-biphosphoglycerate and monophosphoglycerate catalyzed by bisphosphoglycerate phosphatase.

2,3‐Bisphosphoglycerate phosphatase from human erythrocytes was extensively purified with the use of various chromatographic procedures and electrofocusing. All detectable phosphoglyceromutase activity was removed as tested with an optical assay at pH 7.6. At the pH optimum of the phosphatase reaction (pH 6.5) the enzyme transfers phosphate groups from 2,3‐bisphosphoglycerate to monophosphoglycerates (3‐phosphoglycerate or 2‐phosphoglycerate). The transfer is more than hundred times faster than the hydrolyzing activity. Chloride and 2‐phosphoglycolate stimulate the hydrolyzing but not the transferring activity. Data from seven experiments with differently labelled phosphoglycerates were analyzed in terms of rate constants defined by a mathematical model for the different transfer reactions. With the direction of phosphate transfer expressed from 2,3‐bisphosphoglycerate to mono‐phosphoglycerate the constants were estimated to have the following values (ml · min−1·μmol−1): C‐3 → C‐2 0.006 ± 0.001, C‐2 → C‐2 0.024 ± 0.003, C‐3 → C‐3 0.009 ± 0.006 and C‐2 → C‐3 0.034 ± 0.008.The values indicate that the velocity of the transfer from C‐2 in 2,3‐bisphosphoglyerate to the vacant carbon atom in 2‐phosphoglycerate is about the same or possibly somewhat higher than the velocity of the transfer to 3‐phosphoglycerate.The transfer velocity from 2,3‐[3‐32P]bisphosphoglycerate to 3‐phosphoglycerate is much lower. Due to the experimental conditions the results do not allow determination of the velocity of the transfer from C‐3 to 2‐phosphoglycerate with any high degree of precision. The C‐3 to C‐2 transfer however, is slower than the transfer from C‐2 in 2,3‐bisphosphoglycerate.

A model for biological quantum conversion involving the photooxidative dephosphorylation of menadiol diphosphate.

Abstract— The mechanism of the photooxidative dephosphorylation of menadiol diphosphate appears to be invariant with respect to three sensitizers used; namely, riboflavin, biacetyl, and menadiol diphosphate itself. The mechanism involves triplet energy transfer from the sensitizers to oxygen to yield singlet oxygen which oxidizes menadiol diphosphate. The photolysis of menadiol diphosphate in acetic acid has resulted in formation of acetyl phosphate, as determined by paper chromatography. Therefore, we have demonstrated the feasibility of our model for converting light energy into chemical potential (‘high‐energy’ phosphate bond energy or group transfer potential) in the form of acetyl phosphate.