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PubMed1990

Sequencing and enhanced expression of the gene encoding diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae.

The gene, DTP, coding for diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase was isolated from a Saccharomyces cerevisiae genomic DNA library in lambda gt11. In yeast and Escherichia coli transformed with the multicopy vector, YEp352, containing the cloned DTP gene, the Ap4A phosphorylase was produced at levels nine- to 17-fold higher than in untransformed hosts. The nucleotide (nt) sequence was determined. The gene codes for a polypeptide chain of 321 amino acids (aa). Two-aa sequence motifs of possible significance were identified: a potential adenine nt binding site and a potential phosphorylation site. The DTP gene is located on yeast chromosome III and is present as a single copy. Although multicopy vector expression increased the Ap4A phosphorylase activity ninefold above the endogenous activity in transformed yeast, the intracellular concentration of Ap4A did not decrease and the growth rate of the yeast was unchanged.

Amino Acid Sequence↗
PubMed1998

Acceptor specificity of cellobiose phosphorylase from Cellvibrio gilvus: synthesis of three branched trisaccharides.

Cellobiose phosphorylase from Cellvibrio gilvus was examined for its acceptor specificity in the synthetic reaction with glucose-1-phosphate, using substrates in which the C-6 substituent of D-Glc had been altered. A range of disaccharides were also tested for acceptor specificity but only those with (1-->6)-linkages were successful acceptors. Melibiose, gentiobiose, isomaltose and also the monosaccharide glucuronamide were found to react with cellobiose phosphorylase and glucose-1-phosphate giving beta-D-Glcp-(1-->4)-[alpha-D-Galp-(1-->6)]-D-Glcp, beta-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->6)]-D-Glcp, beta-D-Glcp-(1-->4)-[alpha-D-Glcp-(1-->6)]-D-Glcp and beta-D-Glcp-(1-->4)-D-GlcUNp, respectively. These products were purified using a range of chromatographic methods and characterised by NMR and FAB-MS. This is the first time cellobiose phosphorylase has been shown to synthesise trisaccharides.

Carbohydrate Sequence↗
PubMed1998

The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune.

Phosphorolysis of alpha,alpha-trehalose catalyzed by trehalose phosphorylase from the basidiomycete Schizophyllum commune proceeds via net retention of anomeric configuration and yields alpha-D-glucose 1-phosphate and alpha-D-glucose as the products. In reverse reaction, only the alpha-anomers of D-glucose 1-phosphate and D-glucose are utilized as glucosyl donor and acceptor, respectively, and give exclusively the alpha,alpha-product. Trehalose phosphorylase converts alpha-D-glucose 1-fluoride and phosphate into alpha-D-glucose 1-phosphate, a reaction requiring the stereospecific protonation of the glucosyl fluoride by a Brønsted acid. The results are discussed with regard to a plausible reaction mechanism of fungal trehalose phosphorylase.

Glucose↗
PubMed1998

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus.

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30 degrees C. The optimum pH ranges for trehalose degradation and synthesis were 6.0-7.5 and 6.0-7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P(i) (K(i)=2.0 mM). The enzyme was highly specific towards trehalose, P(i), glucose and alpha-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P(i), glucose and alpha-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K(m) values were 61, 4.7, 24 and 6.3 mM for trehalose, P(i), glucose and alpha-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.

Adenosine Triphosphate↗
PubMed2000

Enzymatic synthesis of novel disaccharides using disaccharide phosphorylases.

A method for the synthesis of novel disaccharides was developed. It involved the following two steps. The first step consisted of two continuous reactions: the conversion of maltose to beta-D-glucose-1-phosphate and D-glucose by the phosphorolytic activity of maltose phosphorylase and the specific consumption of only D-glucose by incubation with glucose-consuming yeast cells. The second step involved the addition of an appropriate carbohydrate and its condensation with the remaining beta-D-glucose-1-phosphate by the synthetic activity of maltose phosphorylase or trehalose phosphorylase. Several factors affecting the yields of disaccharides were optimized. Using this method, five maltose-like derivatives and two trehalose-like derivatives were synthesized from maltose and the corresponding carbohydrates. Among these, 4-O-alpha-D-glucopyranosyl-L-fucopyranose (Glc(alpha1-4)Fuc) and alpha-d-glucopyranosyl alpha-D-fucopyranoside (Glc(alpha1-1alpha)Fuc) were purified, and identified by 1H NMR and 13C NMR.

Journal Article↗
PubMed1992

Pyridoxal 5'-phosphate as a 31P reporter observing functional changes in the active site of Escherichia coli maltodextrin phosphorylase after site-directed mutagenesis.

Changes in the active site of Escherichia coli maltodextrin phosphorylase created by substituting residues Lys533, Arg534, Tyr538, and Glu637 were monitored in the absence and presence of arsenate as substrate analogue using pyridoxal-P as 31P NMR reporter. The chemical shift of the cofactor phosphate group of wild-type E. coli phosphorylase is pH dependent with an apparent pK of 5.6 and limiting delta values of 0.71 and 3.6 ppm for the low- and high-pH values, respectively. The apparent pK value of 5.6 indicates that the phosphate group of the cofactor is in hydrogen bond linkage to Lys533. In all mutant enzymes in which the enzymatic activity was significantly reduced, effects on the 31P chemical shift pattern of pyridoxal-P were observed. The K533S, R534Q, E637D, and E637Q mutant enzymes show 0.6, 0.01, 0.2, or 0.1% residual activity, and the apparent pK values of the cofactor phosphate transition of E637D and E637Q mutant enzymes are altered. The Y538F mutant enzyme is a remarkable exception, displaying 12% activity and an environment of the cofactor quite similar to that in wild-type enzyme. This finding suggests that Tyr538, although involved in substrate binding and specificity, is not functionally essential. One crucial aspect of catalysis is the close contact of the phosphates of pyridoxal-P and of substrate rendered by a cluster of positively charged amino acids, Lys533, Lys539, and Arg534. The similar apparent pK values of wild-type and K533S mutant phosphorylase suggest that the cofactor phosphate and the hydroxyl group of Ser533 are linked by a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Arsenates↗
PubMed1988

Synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase.

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.

Adenine Nucleotides↗
PubMed1982

On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus.

The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADP alpha S vs. ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.

Adenosine Diphosphate↗
PubMed2004

Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis.

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and alpha-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B', and C. Domain A comprises the (beta/alpha)(8)-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of beta-sheets 4 and 5 in the (beta/alpha)(8)-barrel, as required for family 13 members. The topology of the B' domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 A(2) per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites.

Amino Acid Sequence↗
PubMed2002

Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis.

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the alpha-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the Km decreased during the proteolytic process when starch was used as the limiting substrate, but the Km for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (Vmax) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis.

Journal Article↗
PubMed2001

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune.

Initial-velocity measurements for the phospholysis and synthesis of alpha,alpha-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before alpha,alpha-trehalose, and alpha-D-glucose is released before alpha-D-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to D-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of D-glucal, a competitive dead-end inhibitor against D-glucose (K(i)=0.3 mM at pH 6.6 and 30 degrees C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2--7.8 and 5.5--6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of k(cat)/K are interpreted in terms of monoanionic phosphate and alpha-D-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The K(i) value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of D-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and alpha-D-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg(2+) that is non-dissociable in the presence of metal chelators. Measurements of the (26)Mg(2+)/(24)Mg(2+) ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

Amino Acid Sequence↗
PubMed2001

Alpha-retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors.

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of alpha,alpha-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme-substrate complexes formed from binary enzyme-phosphate and enzyme-alpha-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and alpha-d-glucopyranosyl phosphate, and binds 3 x 10(4)-fold tighter (K(i) approximately 1 microM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope=1.14; r(2)=0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (K(m)/k(cat))] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log K(i)). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite -1 in the enzyme-phosphate complex with a dissociation constant of 56 microM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of alpha-retaining glucosyltransferase mechanisms that occur with and without a beta-glucosyl enzyme intermediate.

Binding Sites↗
PubMed2002

Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors.

Trehalose phosphorylase is a component of the alpha-D-glucopyranosyl alpha-D-glucopyranoside (alpha,alpha-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from alpha,alpha-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for alpha,alpha-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme-substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (K(i)) for deoxy- and deoxyfluoro derivatives of D-glucose show that loss of interactions with the 3-, 4- or 6-OH, but not the reactive 1- and the 2-OH, results in considerably (> or =100-fold) weaker affinity for sugar-binding subsite +1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the D-glucose-leaving group/nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed alpha-retaining phosphorolysis of alpha-D-glucose-1-fluoride (alpha-D-Glc-1-F) since the productive bonding capability of the fluoride-leaving group with subsite +1 is minimal. The specificity constant (19 M(-1).s(-1)) and catalytic-centre activity (0.1 s(-1)) for the reaction with alpha-D-Glc-1-F are 0.10- and 0.008-fold the corresponding kinetic parameters for the enzymic reaction with alpha,alpha-trehalose. The non-selective-inhibition profile for a series of inactive alpha-D-glycopyranosyl phosphates shows that the driving force for the binary-complex formation lies mainly in interactions of the enzyme with the phosphate group and suggests that hydrogen bonding with hydroxyl groups at the catalytic site (subsite -1) contributes to catalysis by providing stabilization, which is specific to the transition state. Vanadate, a tight-binding phosphate mimic, inhibits the phosphorolysis of alpha-D-Glc-1-F by forming a ternary complex whose apparent dissociation constant of 120 microM is approx. 160-fold greater than the dissociation constant of the same inhibitor complex with alpha,alpha-trehalose.

Arsenates↗
PubMed2007

Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes.

The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

Amino Acid Sequence↗
PubMed1996

Anabaena flos-aquae and other cyanobacteria possess diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) phosphorylase activity.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) phosphorylase, previously only known in lower eukaryotes, has been detected in extracts of the cyanobacteria Anabaena flos-aquae, Anabaena variabilis and Synechococcus sp. The 32 kDa enzyme was partially purified from A. flos-aquae and separated from a 23 kDa hydrolytic activity. It had a pH optimum of 9.5 and required a bivalent cation for activity: Mg2+, Mn2+, Ca2+, Co2+ or Zn2+. Diadenosine tri-, tetra- and penta-phosphates were all effective substrates (relative rates 0.85, 1.00 and 0.27 respectively), while the hexaphosphate was a poor substrate and the diphosphate was inactive. ADP was always one of the products of phosphorolysis. Arsenate and vanadate could substitute for phosphate (relative rates 1.80, 2.25 and 1.00 respectively), but tungstate and sulphate could not. Chromate and molybdate were poor substrates. A search of the GenBank non-redundant database revealed a putative Ap4A phosphorylase gene in the cyanobacterium Synechocystis sp. The gene showed significant blocks of identity/similarity with yeast Ap4A phosphorylases I and II, particularly the latter.

Adenosine Diphosphate↗
PubMed1985

Purification and characterization of polynucleotide phosphorylase from cucumber.

Polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) activity has been found in many prokaryotes and studied in detail since 1955. Such enzymes have been detected also in plants. We now describe the purification of polynucleotide phosphorylase from cucumber cotyledons and leaves. This enzyme is a complex of three subunits, possibly not identical, of about M(r) 50,000. Its enzymatic properties are similar to those of the tobacco enzyme. Unlike the prokaryotic enzymes, the plant enzyme shows activity in the absence of primer but is to various extents stimulated by various ribopolynucleotides or RNAs. RNA-dependent RNA polymerase, not previously shown to exist in non-virus-infected cucumber, has been found to be present at a low level and was separated from the much greater amount of polynucleotide phosphorylase, although some of the physical properties of the two enzymes are rather similar.

Journal Article↗
PubMed1995

An alleged yeast polyphosphate kinase is actually diadenosine-5', 5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase.

Polyphosphates are a major constituent of the yeast Saccharomyces cerevisiae. A purification of the enzyme polyphosphate kinase (E.C. 2.7.4.1) from this organism has been reported (Felter, S., and Stahl, A.J.C. (1973) Biochimie (Paris) 55, 245-251). The assay for activity used in this purification was the production of 32P-labeled nucleotide, presumed to be ATP, in the presence of [32P]polyphosphate and ADP. We have found that this assay does not reflect the activity of a polyphosphate kinase but rather the combination of an exopolyphosphatase, releasing free [32P]phosphate from the added [32P]polyphosphate, and the ADP-[32P]phosphate exchange activity of the enzyme diadenosine 5',5"'-P1, P4-tetraphosphate alpha, beta-phosphorylase (Ap4A phosphorylase). We also present direct evidence for the formation of an enzyme-AMP intermediate in the actin of Ap4A phosphorylase.

Adenosine Monophosphate↗
PubMed1974

'Single addition' and 'transnucleotidation' reactions catalyzed by polynucleotide phosphorylase. Effect of enzymatic removal of inorganic phosphate during reaction.

The reaction of the tetranucleotide, pA-A(2)-A, with 2'(3')-0-(alpha-methoxyethyl)uridine 5'-diphosphate, Mg(2+) ions, and M. luteus polynucleotide phosphorylase followed by mild acid treatment to remove the blocking groups results in a 49% yield of the desired single addition product, pA-A(3)-U, together with smaller amounts of pA-A-U, pA-A-A, pA-A(2)-U, pA-A(2)-A, pA-A(3)-A, pA-A(4)-U, and pA-A(4)-A. The side products are thought to arise from the phosphorolysis of the acceptor molecule by the inorganic phosphate formed in the reaction mixture and from subsequent additions to the various oligonucleotide species by the resulting adenosine 5'-diphosphate. A system developed for the removal of inorganic phosphate as it is formed in the synthesis involves the addition to the reaction mixture of calf spleen nucleoside phosphorylase and nicotinamide riboside and, under these conditions, pA-A(3)-U can be prepared in 90% yield with essentially no side products. Under similar conditions, pA-A(3)-A, pA-A(3)-G, and pA-A(3)-C may be prepared from pA-A(2)-A and the appropriate blocked nucleoside diphosphate in yields of 85-94%. The incubation of pA-A(2)-A alone with polynucleotide phosphorylase exhibits the phenomenon of "transnucleotidation" in that the molecule is partially converted to oligonucleotides of smaller and larger chain lengths. In the presence of the phosphate removal system, however, the tetranucleotide is not attacked by the enzyme, and thus, "transnucleotidation" appears to be simply a combination of phosphorolytic and addition reactions catalyzed by trace amounts of inorganic phosphate contaminating the enzyme and/or the substrate.

Adenine Nucleotides↗