Erythrose 4-phosphate Research Articles

Partial Purification and Characterization of Glucose-6-phosphate Isomerase from Dictyostelium discoideum

SUMMARY: Glucose-6-phosphate isomerase was partially purified from cells of Dictyostelium discoideum in the culmination stage of development. The enzyme had a pH optimum of about 8·5 in Tris/HCl buffer. Activity was not inhibited by p-chloromercuribenzoate or iodoacetate. The enzyme exhibited Michaelis–Menten kinetics and the K m values for glucose 6-phosphate and fructose 6-phosphate were 2 mm and 0·1 mm, respectively. Erythrose 4-phosphate was a strong competitive inhibitor of the enzyme with a K i value of 3·8 μm, whereas 6-phospho-gluconate was less effective with a K i of 0·1 mm.

Formation of 3-octuloses by a self-aldol reaction of d-erythrose

When kept at 105° for 2.5 h, weakly alkaline, syrupy d-erythrose was readily converted into a mixture containing mainly d- glycero-tetrulose, the previously unknown β- d- altro- l- glycero-3-octulofuranose (2), and α- d- gluco- l- glycero-3-octulopyranose, which were isolated as the corresponding acetates. Treatment of 2 with Dowex 50 (H +) resin yielded 3,8-anhydro-β- d- altro- l- glycero-octulopyranose, identified as its acetate. Previous discrepancies in the [α] d values for d-erythrose appear partly to originate in the self-aldol reaction. The dimerisation of d-erythrose 4-phosphate is also described.

Interrelationship between glycolysis and the anaerobic part of the pentose phosphate pathway of carbohydrate metabolism in the myocardium

The reactions of the anaerobic part of the pentose cycle of carbohydrate metabolism and glycolysis in the heart muscle have been compared. In heart muscle the transaldolase activity is either very low or altogether absent; therefore, erythrose 4-phosphate cannot be formed via the transaldolase reaction. The formation of heptulose 1,7-diphosphate from erythrose 4-phosphate and dihydroxyacetone phosphate occurs much faster than fructose 1,6-diphosphate synthesis from triose phosphates. The split-off inorganic phosphate from heptulose 1,7-diphosphate under the action of phosphatase occurs at a higher rate than that from fructose 1,6-diphosphate. The heptulose 7-phosphate formed is an immediate precursor of pentose phosphates in a transketolase reaction with glyceraldehyde 3-phosphate. Hence, during pentose phosphate synthesis from glycolytic products heptulose 1,7-diphosphate after being converted into heptulose 7-phosphate is utilized for the synthesis of pentose phosphates and does not serve as a pool for erythrose 4-phosphate. Under excess of pentose phosphates and the lack of glycolytic products the transketolase reaction between pentose 5-phosphate and erythrose 4-phosphate results in a formation of fructose 6-phosphate and glyceraldehyde 3-phosphate. Thus, erythrose 4-phosphate plays an essential role, i.e., under excess of glycolytic products this process can be channelled into the direction of pentose phosphate formation, while under excess of pentose phosphates — in the direction of synthesis of glycolytic products. The efficient formation of erythrose 4-phosphate occurs via the transketolase reaction between two initial products of glycosis, i.e., glucose 6-phosphate and fructose 6-phosphate to form erythrose 4-phosphate and octulose 8-phosphate. The hexokinase and phosphofructokinase reactions limit the rate of glycolysis; consequently, the retardation of early steps of glycolysis facilitates the participation of glucose 6-phosphate and fructose 6-phosphate in the transketolase reaction. The further pathways of erythrose 4-phosphate utilization are miscellaneous; some of them have been mentioned above. Octulose 8-phosphate can be involved in many reactions, particularly in the transketolase reaction with glyceraldehyde 3-phosphate, resulting in the formation of pentose phosphate and hexose 6-phosphate. Both compounds undergo subsequent conversions depending on the presence and concentration of substrates, which can further be utilized in the reactions of pentose cycle or glycolysis.

Regulation of phospho-2-keto-3-deoxy-heptonate aldolase (DAHP synthase) and anthranilate synthase of Pseudomonas aureofaciens.

Phospho-2-keto-3-deoxy-heptonate aldolase (DAHP synthase) of Pseudomonas aureofaciens ATCC 15926 was inhibited by L-tyrosine. The inhibition was competitive with erythrose 4-phosphate as the varied substrate but non-competitive with respect to phosphoenolpyruvate. Anthranilate synthase was inhibited by L-tryptophan. The inhibition was competitive with respect to chorismate but non-competitive with L-glutamine or NH4+ as the varied substrate. DAHP synthase and anthranilate synthase were not repressed when aromatic amino acids were included in the growth medium. In bacteria grown in the presence of L-phenylalanine, the anthranilate synthase activity was enhanced about threefold compared with the control. Similar results were obtained with the mutant strain P. aureofaciens ACN, which produces increased amounts of pyrrolnitrin.

Functional multiplicity of phosphoglucose isomerase from Lactobacillus casei

Phosphoglucose isomerase ( d-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), purified from Lactobacillus casei, showed multiplicity with respect to electrophoretic mobility, molecular weight, kinetic properties and responses to erythrose 4-phosphate. Among the three forms isolated, one having a dimeric conformation, was specific for glucose 6-phosphate. Erythrose 4-phosphate inhibited this preparation in a sigmoid fashion, while this compound activated the enzyme for isomerization of ribose 5-phosphate. In tetrameric conformation of the similar subunits, the enzyme was more specific for ribose 5-phosphate and the inhibition exerted by erythrose 4-phosphate was hyperbolic. The possible implications of these observations have been discussed.

Effect of heavy water ( 2H 2O) on regulatory properties of phosphoglucose isomerase from Lactobacillus casei

Phosphoglucose isomerase ( d-glucose-6-phosphate ketolisomerase, EC 5.3.1.9) purified to homogeneity from Lactobacillus casei was used for studies on 2H 2O effects. This preparation showed distinct differences in functional properties in H 2O and 2H 2O, respectively. Erythrose 4-phosphate which exerted sigmoidal inhibition in H 2O, acted as a competitive inhibitor in 2H 2O. The enzyme also showed reduced rate of fructose 6-phosphate formation in 2H 2O. The enzyme was found to be dimeric in water and monomeric in 2H 2O. The loss of regulatory properties of the enzyme has been correlated with disaggregation of the protein in heavy water, similar to that caused by sodium dodecyl sulphate treatment.

Effects of inorganic phosphate on the photosynthetic carbon reduction cycle in extracts from the stroma of pea chloroplasts

1. Turnover of the photosynthetic carbon reduction cycle has been demon-strated in chlorophyll-free reaction mixtures containing chloroplast stromal extract, as evidenced by the fixation of CO 2 following addition of small amounts of 3-phosphoglycerate. 2. The activity of the photosynthetic carbon reduction cycle in this system is inhibited by inorganic phosphate (P i), with activity reduced to 50% by about 6.5 mM P i. P i also increased the lag period which elapsed before a steady rate of CO 2 fixation was obtained. 3. The effect of P i on the rate of 3-phosphoglycerate reduction following the addition of substrate amounts of some cycle intermediates was investigated. Substantial inhibition was observed with fructose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and erythrose 4-phosphate as substrates. P i also affected the activity of ribulose-bisphosphate carboxylase, with stimulation at P i concentrations below 2.5 mM and inhibition at higher concentrations. 4. The results showed that the sedoheptulose bisphosphatase reaction is inhibited more strongly by P i than the fructose bisphosphatase reaction. 5. It is concluded that the previously established inhibitory effects of P i on photosynthesis by intact isolated chloroplasts may be partly due to these inhibitory effects of P i on the reactions of the photosynthetic carbon reduction cycle.

Production of glycolate by oxidation of the 1,2-dihydroxyethyl-thamin-diphosphate intermediate of transketolase with hexacyanoferrate(III) or H2O2.

In the presence of hexacyanoferrate(III), or other suitable oxidants, transketolase catalyzes the oxidative cleavage of its donor substrates xylulose 5-phosphate or fructose 6-phosphate into glycolate and glyceraldehyde 3-phosphate or erythrose 4-phosphate, respectively. Two moles of hexacyanoferrate(III) are reduced per mole of oxidatively cleaved donor substrate. In analogy to the oxidative trapping of carbanion intermediates of other enzymes [Healy, M. J. & Christen, P. (1973) Biochemistry, 12, 35-41], the kinetic features of the reaction indicate that the 1,2-dihydroxyethylthiamin diphosphate intermediate is the oxidation-susceptible species. The molecular activity for the oxidative cleavage of fructose 6-phosphate at a hexacyanoferrate(III) concentration of 0.5 mM is 0.2% of that for the normal transfer reaction with erythrose 4-phosphate as acceptor substrate. Glycolate is also produced with H2O2 as oxidant; however, the reaction is at least two orders of magnitude slower than with hexacyanoferrate(III).

Enzymic properties of phosphonic analogues of D-erythrose 4-phosphate

4-Deoxy-D- erythro tetrose 4-phosphonate and 4,5 dideoxy D- erythro pentose 5-phosphonate, the phosphonic analogues of D-erythrose 4-phosphate, have been prepared by oxidation of the corresponding analogues of glucose 6-phosphate and tested as substrates of 3-deoxy-D- arabino heptulosonate 7-phosphate synthetase, transaldolase and transketolase. Kinetic parameters of the reaction with the phosphonate analogues and the natural substrate have been compared.

Detection and estimation of sedoheptulose and octulose mono- and bisphosphates in extracts of rat liver

Sedoheptulose 1,7-bisphosphate has been shown to be present in extracts of normal rat liver. Its concentration in this tissue, estimated by colorimetric and enzymatic assays, is in the range of 5–7 nmol/g tissue. The concentration of sedoheptulose 7-phosphate in these extracts was 110 nmol/g tissue. Also present were mono- and bisphosphate esters of d- glycero- d- ido-octulose and d- glycero- d- altro-octulose, in concentrations ranging from 1–10 nmol/g tissue. Sedoheptulose 1,7-bisphosphate may function as a reservoir for erythrose 4-phosphate. The possible origin of the eight-carbon sugars and their function are discussed.

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.

An enzymic method for the analysis of d-erythrose 4-phosphate

Two modifications of an enzymic method for the measurement of erythrose 4-phosphate, based on reactions catalyzed by transketolase, are described. Unlike the method generally employed, the new procedure is sensitive and highly specific and applicable to the assay of erythrose 4-phosphate in tissue extracts.

Iron, an essential element for biosynthesis of aromatic compounds.

Homogeneous preparations of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.1.2.15] isolated as the enzyme-phosphoenolpyruvate complex from Escherichia coli are shown by atomic absorption analysis to contain approximately one mole of iron per mole of native enzyme. No cobalt was found, in contrast to suggestions of earlier workers. Pure enzyme preparations show a unique absorption maximum around 350 nm with an epsilon value of about 3500 M-1cm-1. The 350-nm band as well as the enzyme activity is lost when the enzyme is denatured with guanidine-hydrochloride, or when phosphoenolpyruvate, the first substrate to bind to the enzyme, is totally removed from the enzyme by incubation with an excess of erythrose 4-phosphate, the second substrate to bind to the enzyme. The iron remains bound to the enzyme when phosphoenolpyruvate is removed from the enzyme-phosphoenolpyruvate complex.

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.

Fluorine-containing analogues of intermediates in the Shikimate pathway.

The phosphoenolpyruvate analogue (Z)-phosphoenol-3-fluoropyruvate is a substrate for phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase from Escherichia coli. In the presence of excess erythrose 4-phosphate, apparent KM values of 65 and 38 muM were observed for phosphoenol-3-fluoropyruvate and phosphoenolpyruvate, respectively. Because the apparent Vmax for phosphoenol-3-fluoropyruvate is only 1.17% of that for phosphoenolpyruvate, one can study the former as an inhibitor of 3-deoxy-arabino-heptulosonic acid-7-phosphate synthase. Kinetic experiments showed phosphoenol-3-fluoropyruvate to be competitive with respect to phosphoenolpyruvate. Two distinguishable Ki values of 8 and 48 muM were obtained. The product (3S)-3-deoxy--3-fluoro-arabino-heptulosonic acid 7-phosphate was purified, characterized, and shown to act as a substrate for 5-dehydroquinate synthase. 3-Deoxy-3-fluoro-arabino-heptulosonic acid 7-phosphate, in contrast to 3-deoxy-arabino-heptulosonic acid 7-phosphate reacts slowly or not at all with reagents specific for 2-keto-3-deoxy sugars and is relatively resistant to oxidative cleavage by sodium periodate. The expected product of periodate oxidation, 3-fluoro-3-formylpyruvate, cannot be detected. This observation was clarified by studies with model compounds.

Dimerization of erythrose 4-phosphate

Dimerization of erythrose 4-phosphate

Half-of-the-sites activity of transaldolase: Titration of the active site and characteristics of the slow reaction catalyzed by the second subunit

When transaldolase is incubated with the donor substrate fructose 6-phosphate in the absence of the acceptor substrate erythrose 4-phosphate, there is a very rapid release of glyceraldehyde 3-phosphate, approaching a ratio of 1 mol/mol of enzyme within 1 min and corresponding to the formation of 1 mol of dihydroxyacetone-enzyme intermediate. This burst reaction is proportional to the amount of enzyme present and takes place in the pH range of the enzymatic activity. It is inhibited by phosphate, which is a competitive inhibitor of fructose 6-phosphate in the enzymatic transfer reaction. Following the burst reaction, there is a slow reaction of the enzyme with fructose 6-phosphate with a k cat 1000 times less than that of the complete reaction. This slow reaction, but not the burst, is also observed with the reduced dihydroxyacetone-transaldolase intermediate, which is inactive in the complete reaction. The K m for fructose 6-phosphate is in the same range for both reactions. Transaldolase is a dimer composed of identical subunits, and the data indicate that it is a half-site enzyme exhibiting almost complete negative cooperativity. If one active site is blocked by reduction of the Schiffs base intermediate, the second site still catalyzes the slow cleavage of fructose 6-phosphate. The rate of this slow reaction reflects the extent of the negative cooperativity.

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.

Mechanistic implications of the pH independence of inhibition of phosphoglucose isomerase by neutral sugar phosphates.

In contrast to the strongly pH-dependent inhibition of phosphoglucose isomerase by substrate analogues with a free carboxyl group, inhibition of this enzyme by neutral sugar phosphates is essentially invariant between pH 7 and 9. Competitive inhibition constants for glucitol 6-phosphate (40 muM), arabinose 5-phosphate (50 muM), and erythritol 4-phosphate (100 muM) were found to be of the same order of magnitude as that reported previously for substrate binding constants (50 to 240 muM). The unique exception is erythrose 4-phosphate whose Ki (0.7 muM, independent of pH) reflects a tightness of binding similar to that found at pH values near or below neutrality for the transition state analogue 5-phosphorarabinonate. The pH independence of inhibition by erythrose 4-phosphate and other neutral sugar phosphates may reflect a mode and locus of binding to phosphoglucose isomerase different from that of the aldonate inhibitors.

The synthesis of 3-deoxyheptulosonic acid 7-phosphate.

3-Deoxyheptulosonic acid 7-phosphate was obtained by a one-step chemical synthesis through condensation of oxalacetate with erythrose 4-phosphate. This reaction occurs at measurable rates only in the presence of a metal ion; Co2+ and Ni2+ are the most effective catalysts. The Co2+ catalyzed condensation of oxalacetate and erythrose 4-phosphate proceeds at room temperature and neutral pH. Since erythrose 4-phosphate can be replaced by any free aldehyde tested thus far, this type of a homogeneous catalysis opens new synthetic routes to alpha-keto-gamma-hydroxy-fatty acids and their derivatives.