Synthesis of the coenzymes adenosine diphosphate glucose,guanosine diphosphate glucose,and cytidine diphosphoethanolamine under primitive earth conditions

The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.     

Trehalose phosphate synthesis in Streptomyces hygroscopicus: purification of guanosine diphosphate D-glucose: D-glucose-6-phosphate 1-glucosyl-transferase

Guanosine diphosphate d-glucose:d-glucose-6-phosphate 1-glucosyl-transferase was purified approximately 100-fold from extracts of Streptomyces hygroscopicus. The purified enzyme catalyzed the transfer of glucose from guanosine diphosphate-d-glucose to glucose-6-phosphate to form trehalose phosphate and guanosine diphosphate. The enzyme was specific for these two substrates and was stimulated by the addition of magnesium ions. The product was characterized as alpha-alpha-trehalose-6-phosphate by its physical and chemical properties. The enzyme was present in a large number of Streptomyces species, suggesting that this group of organisms synthesized trehalose phosphate in a unique manner. This enzyme was not detected in fungi, since these organisms utilized uridine diphosphate-d-glucose as the glucosyl donor.     

Cellulose synthesis by acetobacter xylinum II. Investigation into the relation between cellulose synthesis and cell envelope components

Cell envelope fractions, capable of cellulose synthesis from uridine diphosphate glucose, α-glucose-1-phosphate, glucose-6-phosphate and glucose, have been isolated from Acetobacter xylinum suspensions and various enzymatic properties examined.Essential enzymes were found to be distributed throughout the cell envelope region, with both inner (cytoplasmic) and outer (cell wall) membranes contributing to cellulose synthesis. The central role of UDPG in cellulose synthesis was confirmed and the results indicated that the nucleoside diphosphate sugar functions solely in the cell envelope region of whole cells. A comparison of properties of the cell envelope system with those of different preparations used by other workers, suggested that the method of cell disruption may influence substrate specificity.     

Changes in 32P-phosphorus compounds during translocation in Laminaria digitata (L.) lamouroux

³²P was applied to a Laminaria digitata thallus and the pattern of ³²P phosphorylated compounds was studied, as a function of time, in the different tissues involved in translocation, i.e. source, pathway and sinks. The results showed that, 3 hours after absorption by the uptake region (lamina), the bulk of the radioactivity was incorporated into organic compounds (70 to 80% of total ³²P taken up), hexose monophosphates being the heaviest labelled. Further change in that region was marked by an accumulation of ³²P in the inorganic pool (65 to 70% after 13 days). Conversely, the ³²P pattern in the medulla of the stipe, which initially showed a similar pattern to the uptake region, did not vary during translocation. The pattern of ³²P distribution into sinks (growing stipe peripheral tissue or hapteron) leads to accumulation of the radioactive element in inorganic and acid-insoluble fractions. These results are discussed in terms of comparative distribution of ³²P in the different parts of the thallus and suggest that phosphate moves as Pi in that alga.Abbreviations TCA trichloroacetic acid - Po organic phosphate - Po sol acid-soluble organic phosphate fraction - Po insol acidinsoluble organic phosphate fraction - Pi morganic phosphate fraction - P lip lipidic phosphate - Np protein nitrogen - ATP adenosine triphosphate - ADP adenosine diphosphate - PEP phosphoenolpyruvic acid - PGA phosphoglyceric acid - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - UDPG uridine diphosphoglucose     

Effects of Pyrophosphate on Formation of Nucleotide Derivatives

It was found that CDP-choline was formed with good yield from 5′-CMP and choline phosphate or choline chloride by yeast cells. The effects of pyrophosphate (PP_i) on the formation of UDPG, GDPM and CDP-choline from respective nucleoside monophosphate by yeast cells were studied. By the addition of PP_i to the reaction mixture, the phosphorylation of G-6-P from glucose was inhibited and then the phosphorylation of nucleoside monophosphates was restrained. Such inhibition was reversed by the decomposition of PP_i by inorganic pyrophosphatase of yeast cells. The addition of PP_i after the formation of nucleotide derivatives caused the accumulation of UTP and GTP and molar yields from nucleotide as substrate was about 80%. But that of CTP was a little in the reaction system of CDP-choline synthesis. Further, this method seems to be suitable for the accumulation of sugar-1-phosphates.     

Effects of catecholamine treatment on UDPG levels in white and red skeletal muscle

Adrenaline causes of 5 fold increase of glucose-6-phosphate and of glucose-1-phosphate both in the white extensor digitorum longus and in the red soleus (where the levels of the two sugar phosphates are significantly lower). On the other hand, UDPG levels--which are similar in the two muscles--are significantly decreased after adrenaline. It has been concluded that the levels of glucose-1-phosphate and of UDPG in muscle are not bound to change together.     

SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (P(i)). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i)-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i) exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i)-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Rat liver glucose-6-phosphatase system: light scattering and chemical characterization

Glucose-6-phosphatase is a multicomponent system located in the endoplasmic reticulum, involving both a catalytic subunit (G6PC) and several substrate and product carriers. The glucose-6-phosphate carrier is called G6PT1. Using light scattering, we determined K(D) values for phosphate and glucose transport in rat liver microsomes (45 and 33mM, respectively), G6PT1 K(D) being too low to be estimated by this technique. We provide evidence that phosphate transport may be carried out by an allosteric multisubunit translocase or by two distinct proteins. Using chemical modifications by sulfhydryl reagents with different solubility properties, we conclude that in G6PT1, one thiol group important for activity is facing the cytosol and could be Cys(121) or Cys(362). Moreover, a different glucose-6-phosphate translocase, representing 20% of total glucose-6-phosphate transport and insensitive to N-ethylmaleimide modification, could coexist with liver G6PT1. In the G6PC protein, an accessible thiol group is facing the cytosol and, according to structural predictions, could be Cys(284).     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Adenosine Triphosphate and Other Requirements for the Utilization of Glucose by Agents of the Psittacosis-Trachoma Group.

Weiss, Emilio (Naval Medical Research Institute, Bethesda, Md.). Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-trachoma group. J. Bacteriol. 90:243-253. 1965.-The agent of meningopneumonitis cultivated in the allantoic cavity of chick embryos and purified by differential centrifugations was employed for most of the studies of the requirements for glucose utilization. The evolution of C(14)O(2) from glucose-1-C(14) was used as the criterion of metabolic activity in most experiments. The rate of glucose utilization increased somewhat during the first hour of incubation at 34.4 C and became approximately constant during the second hour. Changes in glucose concentration from 1 to 5 mm did not appreciably affect metabolic activity. More vigorous CO(2) production was obtained when the ratio of K(+)-Na(+) was >1 and, under certain conditions, when the concentration of inorganic phosphate was relatively high (0.05 m). Glucose utilization was entirely dependent on added adenosine triphosphate (ATP) and Mg(++). The effect of ATP was greatly reduced when the microorganisms were partially disrupted with sonic energy. Adenosine diphosphate (ADP) could be substituted for ATP, but the activity was reduced to less than 20%. ATP was not required when glucose-6-phosphate was substituted for glucose. With ADP and glucose, glucose-6-phosphate was an effective competitor of glucose utilization. Nicotinamide adenine dinucleotide phosphate (NADP) enhanced CO(2) production from carbon 1, but not from other carbons, with glucose and, especially, glucose-6-phosphate as substrates. ATP and NADP produced the above-described effects only when their concentrations were comparable to those of the substrates. These concentrations always exceeded the amount of CO(2) produced (0.05 to 0.5 mumole/mg of agent protein). The concentration of NADP could be reduced when oxidized glutathione was added. Diphosphothiamine had no effect on CO(2) production. Qualitatively similar results were obtained with the agent of trachoma purified from yolk sac. These experiments furnish evidence that agents of the psittacosistrachoma group, despite their enzymatic capabilities, require an exogenous source of energy.     

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta.

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6P and beta-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate: glucose phosphotransferase, and undetectable [U-14C]G6P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate/pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.     

Identification and characterisation of a new human glucose-6-phosphatase isoform

The liver endoplasmic reticulum glucose-6-phosphatase catalytic subunit (G6PC1) catalyses glucose 6-phosphate hydrolysis during gluconeogenesis and glycogenolysis. The highest glucose-6-phosphatase activities are found in the liver and the kidney; there have been many reports of glucose 6-phosphate hydrolysis in other tissues. We cloned a new G6Pase isoform (G6PC3) from human brain encoded by a six-exon gene (chromosome 17q21). G6PC3 protein was able to hydrolyse glucose 6-phosphate in transfected Chinese hamster ovary cells. The optimal pH for glucose 6-phosphate hydrolysis was lower and the K(m) higher relative to G6PC1. G6PC3 preferentially hydrolyzed other substrates including pNPP and 2-deoxy-glucose-6-phosphate compared to the liver enzyme.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Expression of melanoma neutral proteinase and collagenase potential by endocytosis.

When oleoyl phosphate and ADP were incubated with heart submitochondrial particles in the presence of glucose-hexokinase trap according to a reported procedure [Griffiths, D.E. (1976) Biochem. J. 160: 809–812], a 10% yield of glucose-6-phosphate was detected by chemical analysis. Although lower concentration of oleoyl phosphate improved the yield to 80–85%, the mode of formation of glucose-6-phosphate was not clear under the experimental condition used to improve the yield. In order to test decisively whether the phosphoryl group of oleoyl phosphate was transferred to ADP to form ATP which was estimated in the form of glucose-6-phosphate, [³²P]oleoyl phosphate was synthesized. The use of isotopically labelled oleoyl phosphate showed only about 5% yield of [³²P]glucose-6-phosphate by paper chromatographic analysis, whereas chemical analysis of the same system gave 80% yield of glucose-6-phosphate. Such an observation demonstrated that glucose-6-phosphate estimated by chemical assay is not the result of phosphorylation of ADP with oleoyl phosphate catalyzed by the submitochondrial particles.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

The molecular basis of type 1 glycogen storage diseases

Glycogen storage disease type 1 (GSD-1), also known as von Gierke disease, is a group of autosomal recessive metabolic disorders caused by deficiencies in the activity of the glucose-6-phosphatase (G6Pase) system that consists of at least two membrane proteins, glucose-6-phosphate transporter (G6PT) and G6Pase. G6PT translocates glucose-6-phosphate (G6P) from cytoplasm to the lumen of the endoplasmic reticulum (ER) and G6Pase catalyzes the hydrolysis of G6P to produce glucose and phosphate. Therefore, G6PT and G6Pase work in concert to maintain glucose homeostasis. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Both manifest functional G6Pase deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-1b patients also suffer from chronic neutropenia and functional deficiencies of neutrophils and monocytes, resulting in recurrent bacterial infections as well as ulceration of the oral and intestinal mucosa. The G6Pase gene maps to chromosome 17q21 and encodes a 36-kDa glycoprotein that is anchored to the ER by 9 transmembrane helices with its active site facing the lumen. Animal models of GSD-1a have been developed and are being exploited to delineate the disease more precisely and to develop new therapies. The G6PT gene maps to chromosome 11q23 and encodes a 37-kDa protein that is anchored to the ER by 10 transmembrane helices. A functional assay for the recombinant G6PT protein has been established, which showed that G6PT functions as a G6P transporter in the absence of G6Pase. However, microsomal G6P uptake activity was markedly enhanced in the simultaneous presence of G6PT and G6Pase. The cloning of the G6PT gene now permits animal models of GSD-1b to be generated. These recent developments are increasing our understanding of the GSD-l disorders and the G6Pase system, knowledge that will facilitate the development of novel therapeutic approaches for these disorders.     

Glycogen synthase Hymenolepis diminuta. I. Allosteric activation and inhibition.

Glycogen synthase (UDP glucose: glycogen alpha-4-glycosyltransferase, EC2.4.1.11) of the tapeworm Hymenolepis diminuta exists in 2 forms: 1) the I-form (independent), which has significant activity in the absence of glucose 6-phosphate (G6P); and 2) the phosphorylated D-form (dependent), which has no enzymatic activity unless G6P is present. The activity of the I-form is greatly enhanced by a variety of allosteric effectors which have, as their common feature, 1 or more phosphate groups. These include inorganic phosphate (Pi), several sugar phosphates, some phosphorylated glycolytic intermediates, and nucleoside mono- and triphosphates. Competition studies suggest that while most of the positive effectors act at the same site on the enzyme (the "G6P site"), fructose 1,6-diphosphate (FDP) and 2,3-diphosphoglyceric acid (2,3DPG) act at low concentrations to stimulate the enzyme at another locus (the "diphosphate site"), while at high concentrations they competitively inhibit the binding of G6P and of the other activators. The inhibition by high uridine monophosphate (UMP) concentrations is competitive only with the activator uridine triphosphate (UTP), suggesting the existence of a third type of allosteric site (the "uridine nucleotide site"). This third site may be the locus for feedback inhibition by the product uridine diphosphate (UDP), a control mechanism which has been observed to occur in mammalian systems. The allosteric control of the D-form of the enzyme is comparatively simple, apparently involving only one site (the "G6P site") that binds a few effects with greatly reduced affinity. Pi reverses the activation of the D-form by G6P.