Legionella pneumophila Is Directly Sensitive to 2-Deoxyglucose-Phosphate via Its UhpC Transporter but Is Indifferent to Shifts in Host Cell Glycolytic Metabolism.

Toll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection with Legionella pneumophila, but the role of host macrophage glycolysis in terms of intracellular L. pneumophila replication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocks L. pneumophila replication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG on L. pneumophila in macrophages requires the L. pneumophila hexose-phosphate transporter UhpC. Experiments with UhpC-deficient L. pneumophila revealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG on L. pneumophila in mammalian cells requires 2DG phosphorylation. UhpC-deficient L. pneumophila replicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellular L. pneumophila replication. These results are consistent with the view that L. pneumophila can employ diverse metabolic strategies to exploit its host cells.IMPORTANCE We explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen, Legionella pneumophila Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication of L. pneumophila during infection of macrophages, leading to speculation that L. pneumophila may exploit macrophage glycolysis. We isolated L. pneumophila mutants resistant to the inhibitory effect of 2DG in macrophages, identifying a L. pneumophila hexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication of L. pneumophila, which is consistent with a view of L. pneumophila as a metabolic generalist.

Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

Inhibition of SnRK1 by metabolites: tissue-dependent effects and cooperative inhibition by glucose 1-phosphate in combination with trehalose 6-phosphate.

SnRK1 of the SNF1/AMPK group of protein kinases is an important regulatory protein kinase in plants. SnRK1 was recently shown as a target of the sugar signal, trehalose 6-phosphate (T6P). Glucose 6-phosphate (G6P) can also inhibit SnRK1 and given the similarity in structure to T6P, we sought to establish if each could impart distinct inhibition of SnRK1. Other central metabolites, glucose 1-phosphate (G1P), fructose 6-phosphate and UDP-glucose were also tested, and additionally ribose 5-phosphate (R5P), recently reported to inhibit SnRK1 strongly in wheat grain tissue. For the metabolites that inhibited SnRK1, kinetic models show that T6P, G1P and G6P each provide distinct regulation (50% inhibition of SnRK1 at 5.4 µM, 480 µM, >1 mM, respectively). Strikingly, G1P in combination with T6P inhibited SnRK1 synergistically. R5P, in contrast to the other inhibitors, inhibited SnRK1 in green tissues only. We show that this is due to consumption of ATP in the assay mediated by phosphoribulokinase during conversion of R5P to ribulose-1,5-bisphosphate. The accompanying loss of ATP limits the activity of SnRK1 giving rise to an apparent inhibition of SnRK1. Inhibition of SnRK1 by R5P in wheat grain preparations can be explained by the presence of green pericarp tissue; this exposes an important caveat in the assessment of potential protein kinase inhibitors. Data provide further insight into the regulation of SnRK1 by metabolites.

Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase.

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

Glucose and its metabolites have distinct effects on the calcium-induced mitochondrial permeability transition.

Mitochondrial production of reactive oxygen species (ROS) due to up-regulated glucose oxidation is thought to play a crucial, unifying role in the pathogenesis of chronic complications associated with diabetes mellitus. Mitochondrial permeability transition (MPT) is an interesting phenomenon involved in calcium signalling and cell death. We investigated the effects of glucose and several of its metabolites on calcium-induced MPT (measured as mitochondrial swelling) in isolated rat liver mitochondria. The presence of glucose, glucose 1-phosphate (both at 30 mM) or methylglyoxal (6 mM) significantly slowed calcium-induced mitochondrial swelling. Thirty mM glucose also resulted in a significant delay of MPT onset. In contrast, 30 mM fructose 6-phosphate accelerated swelling, whereas glucose 6-phosphate did not influence the MPT. The calcium binding potentials of the three hexose phosphates were tested and found similar. In vitro hydrogen peroxide production by mitochondria respiring on succinate in the presence of rotenone was independent of mitochondrial membrane potential and increased transiently during calcium-induced MPT. Inhibition of MPT with cyclosporine A resulted in decreased mitochondrial ROS production in response to calcium. In contrast, inhibition of MPT by methylglyoxal was accompanied by increased ROS production in response to calcium. In conclusion, we confirm that methylglyoxal is a potent inhibitor of MPT. In addition, high levels of glucose, glucose 1-phosphate and fructose 6-phosphate can also affect MPT. Methylglyoxal simultaneously inhibits MPT and increases mitochondrial ROS production in response to calcium. Our findings provide a novel context for the role of MPT in glucose sensing and the cellular toxicity caused by methylglyoxal.

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU.

GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-ß-d-glucopyranoside 6-phosphate (1ß), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1ß, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1ß and 2 were inactive against GlmM and GlmU even at high concentrations.

Glucose 1-phosphate increases active transport of calcium in intestine.

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.

Effect of diluents on motility of ram sperm during storage at 5 degrees C.

This study examined the effects of 5 diluents--glucose-phosphate (GP), skim milk-egg yolk (MY), egg yolk-citrate (YC), blood sera (S), and seawater (SW)--on daily motility of ram sperm stored at 5 degrees C for a short time. Ram semen collected by artificial vagina was diluted. Percentage of motile spermatozoa (PM) was assessed every 24 h up to day 10 after dilution. The different kinds of diluents except for SW had no effect on the primary motility of sperm and the primary motility was 0 with SW. There was a significant effect (p<.01) of diluent type and storage times in the conservation rates after cooling for PM. The GP diluent achieved greater values (80.5+/-0.88%) than S (69.5+/-1.70%) and MY (78.0+/-1.17%), while YC showed an intermediate value (79.0+/-1.24%). In relation to time of storage, GP and YC diluents showed the PM values up to day 10, but these values were 8 and 7 days for MY and S, respectively. The GP and YC extenders maintained a PM > 50% up to day 4, while MY was greater than 50% up to day 3 and S to day 2.

The in vitro effects of galactose and its derivatives on rat brain Mg2+-ATPase activity.

Galactosaemia is an inborn error of metabolism characterized by irreversible damage to neural tissue. To evaluate whether galactose metabolic disorders, (e.g. classical galactosaemia, galactokinase deficiency galactosaemia), is implicated for alterations of brain Mg2+-ATPase activity, various concentrations (1-16 mM) of galactose, galactose-1-phosphate, galactitol, glucose-1-phosphate or glucose were preincubated with whole brain homogenates of suckling rats at 37 degrees for 1 hr. Mg2+-ATPase activities were determined according to Bowler & Tirri's (1974). Galactose-1-phosphate or glucose-1-phosphate excessively activated the brain Mg2+-ATPase in a concentration-dependent way. Additionally, galactitol, galactose or glucose stimulated the enzyme up to 35-45% (P < 0.001) at concentrations >4 mM. A mixture of galactose-1-phosphate (2 mM), glactitol (2 mM) and galactose (4 mM), concentrations commonly found in blood and brain of untreated patients with classical galactosaemia, resulted in a 500% enzyme activation (P < 0.001) as compared to control. Moreover, a mixture of galactitol (2 mM) and galactose (1 mM), concentrations measured in patients with galactokinase deficiency, caused an enzyme stimulation (35%, P < 0.001). These findings suggest: a) The great Mg2+-ATPase activation by galactose-1-phosphate or glucose-1-phosphate may be due to the epimer of galactose and the presence of phosphorus. b) The brain Mg2+-ATPase stimulation by galactose and its derivatives could be toxic by modulating the Mg2+ concentration, the ATP availability, the activity of other ATP- and Mg2+-dependent enzymes as well as the rates of protein synthesis and cell growth.

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.

Simplification of adenophostin A defines a minimal structure for potent glucopyranoside-based mimics of D-myo-inositol 1,4,5-trisphosphate.

The synthesis of 1-O-[(3S,4R)-3-hydroxytetrahydrofuran-4-yl]-alpha-D-glucopyranosid e 3,4,3'-trisphosphate (7), a novel Ca2+ mobilising agonist at the Ins(1,4,5)P3 receptor, designed by excision of two motifs of adenophostin A is reported, defining a potential minimal structure for potent glucopyranoside-based agonists of Ins(1,4,5)P3 receptors.

Purification and properties of mycobacterial GDP-mannose pyrophosphorylase.

The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis. At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP[32P]-d-mannose. The purified enzyme was stable for several months when kept in the frozen state. The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae. In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity. Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate. The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate. ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive. The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme. The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM. This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides.

Molecular analysis of the interaction between the Bacillus subtilis trehalose repressor TreR and the tre operator.

The trehalose operon of Bacillus subtilis is subject to regulation by induction, mediated by the repressor TreR, and by carbon catabolite repression (CCR). For in vitro investigations, TreR from B. subtilis was overproduced and purified. Its molecular mass, as estimated by SDS-PAGE, is 27 kDa. Size fractionation under native conditions yielded a size estimate of 56 kDa, indicating that TreR exists as a dimer in its native state. Analysis of its interaction with various DNA fragments shows that TreR is able to recognize two tre operators with different efficiencies, and indicates cooperative binding. Previous results have suggested that CCR of the tre operon occurs by a mechanism in which the specific regulator, TreR, may be involved independently of the central component, CcpA. The data presented here indicate that the TreR-tre operator interaction is influenced by several effectors. Thus, the presence of trehalose-6-phosphate, as well as glucose-1-phosphate and sodium chloride, inhibits tre operator binding. Glucose-6-phosphate can act as an anti-inducer, which might reflect its additional role in CCR exerted by glucose.

Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase.

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5, 5'-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.

Fatty acid binding to rat liver fatty acid-binding protein is modulated by early glycolytic intermediates.

Fatty acid binding to rat liver fatty acid binding protein in the presence of glycolytic metabolites and at different pH (optimal 7.2) and ionic strength was studied. Binding decreased logarithmically with ionic strength. Glucose and glucose-6-phosphate increased fatty acid binding significantly with K0.5 within physiological ranges while glucose-1-phosphate and phosphate ion caused no effect.

[Synthesis and biological properties of 3,5-cyclophosphates- and -amidophosphates of 1,2-O-alkylydene-6-deoxy-6-halogeno-alpha -D-glucofuranoses]. / Sintez i biologicheskie svoistva 3,5-tsiklofosfatov i amidofosfatov 6-galoid-6-dezoksi-1,2-O-alkiliden-alpha-D-gliukofuranoz.

3,5-Cyclic phosphates and phosphoramides of 6-halogenated glucofuranoses were synthesized via interaction of 3,5,6-bicyclophosphites of 1,2-O-alkylidene-alpha-D-glucofuranoses with halogens (followed by treatment with nucleophilic reagents) and N-chloroamines. 3,5-Cyclic trans-dibutylphosphoramides of 6-chloro-6-deoxy-1,2-O-isopropylidene- and 6-chloro-6-deoxy-(R)-(2,2,2)-trichloroethylidene)-alpha-D-glucofuranoses were shown to possess antiproliferative activity against CaOv human ovarian carcinoma cells in vitro (CE50 of approximately 10(-5) M). Cyclic trans-dibutylphosphoramide of 6-chloro-6-deoxy-1,2,-O-isopropylidene-alpha-D-glucofuranose also displayed marked antitumor effect on P-388 transplantable murine leukemia in vivo (the maximum increase in life span of 100% was reached at the quintuple injection of 100 mg/kg daily).

A protein phosphorylation switch at the conserved allosteric site in GP.

A phosphorylation-initiated mechanism of local protein refolding activates yeast glycogen phosphorylase (GP). Refolding of the phosphorylated amino-terminus was shown to create a hydrophobic cluster that wedges into the subunit interface of the enzyme to trigger activation. The phosphorylated threonine is buried in the allosteric site. The mechanism implicates glucose 6-phosphate, the allosteric inhibitor, in facilitating dephosphorylation by dislodging the buried covalent phosphate through binding competition. Thus, protein phosphorylation-dephosphorylation may also be controlled through regulation of the accessibility of the phosphorylation site to kinases and phosphatases. In mammalian glycogen phosphorylase, phosphorylation occurs at a distinct locus. The corresponding allosteric site binds a ligand activator, adenosine monophosphate, which triggers activation by a mechanism analogous to that of phosphorylation in the yeast enzyme.