Competitive inhibition of Trypanosoma brucei phosphoglucose isomerase by D-arabinose-5-phosphate derivatives

We report four new strong high energy intermediate analog competitive inhibitors of fructose-6-phosphate isomerization catalyzed by purified Trypanosoma brucei phosphoglucose isomerase: D-arabinonhydroxamic acid-5-phosphate, D-arabinonate-5-phosphate, D-arabinonamide-5-phosphate and D-arabinonhydrazide-5-phosphate. For comparison, the inhibitory properties of the corresponding non-phosphorylated analogues D-arabinonhydroxamic acid, D-arabinonate, D-arabinonamide and D-arabinonhydrazide were also evaluated. D-Arabinonhydroxamic acid-5-phosphate appears as the most potent competitive inhibitor ever evaluated on a phosphoglucose isomerase with an inhibition constant value of 50 nM and a Michaelis constant over inhibition constant ratio of about 2000. Our results show that anionic high energy intermediate analogues, and more particularly D-arabinonhydroxamic acid-5-phosphate, display a weak but significant specificity for Trypanosoma brucei phosphoglucose isomerase versus yeast phosphoglucose isomerase, while neutral high energy intermediate analogues are not selective at all. This would indicate the presence of more positively charged residues in the active site for Trypanosoma brucei phosphoglucose isomerase as compared to that of yeast phosphoglucose isomerase.     

The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition

Phosphoglucose isomerase catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. In addition, phosphoglucose isomerase has been shown to have functions equivalent to neuroleukin, autocrine motility factor, and maturation factor. Here we present the crystal structures of phosphoglucose isomerase complexed with 5-phospho-D-arabinonate and N-bromoacetylethanolamine phosphate at 2.5- and 2.3-A resolution, respectively. The inhibitors bind to a region within the domains' interface and interact with a histidine residue (His(306)) from the other subunit. We also demonstrated that the inhibitors not only affect the enzymatic activity of phosphoglucose isomerase, but can also inhibit the autocrine motility factor-induced cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible isomerization mechanism for phosphoglucose isomerase.     

Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.     

Phenotypes of Phosphoglucose isomerase,phosphoglucose mutase and general protein in some freshwater molluscs from Basrah,Iraq

Electrophoretic variants of phosphoglucose isomerase (EC.5.3.1.9) and phosphoglucose mutase (EC.2.7.5.1) have been studied in eight species of freshwater molluscs. Two phenotypes of phosphoglucose isomerase were observed in Melanopsis nodosa and one phenotype was observed in the rest of the species. One phenotype of phosphoglucose mutase was observed in all the species of molluscs studied. Phosphoglucose isomerase is inferred to be a dimer encoded at a single polymorphic locus in Melanoides nodosa. There are two alleles at this locus. Phosphoglucose mutase is inferred to be a monomer encoded at a single monomorphic locus in all species. The electrophoretic analysis revealed that phosphoglucose isomerase enzyme cannot be considered a good taxonomic criterion to differentiate the different members of the six families studied but, on the other hand, it is considered a good taxonomic criterion to differentiate Melanopsis nodosa and Theodoxus jordani. Phosphoglucose mutase is considered a good taxonomic criterion to differentiate the family Melanidae from the remaining five families studied. General protein can be considered a good taxonomic criterion to differentiate the family Corbicullidae from Melanidae, Viviparidae and Neritidae but, on the other hand, it seems to be a less useful taxonomic criterion to differentiate between the Viviparidae and Neritidae.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

A New Role of Phosphoglucose Isomerase. Involvement of the Glycolytic Enzyme in Aldehyde Metabolism

Lipid peroxidation in biological membranes is accompanied by malonic dialdehyde (MDA) formation, but the problem of its further metabolism in cytoplasm remains unsolved. The experimental data obtained in this work showed that the liver fraction prepared by centrifugation at 10,000g contained phosphoglucose isomerase and enzymes of the glyoxalase system. In this fraction in the presence of GSH there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes (MDA conversion to methylglyoxal and further to neutral D-lactate). This means that MDA is slowly accumulated because it is a substrate of aldehyde isomerase (MDA <--> methylglyoxal). Most probably, phosphoglucose isomerase serves as this enzyme. We concluded that D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.     

Cumene peroxide and Fe2+-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase

Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA ↔ methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 ± 8 μM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Biochemical genetic comparison of the Chinese and European races of the common carp

An electrophoretic survey of the common carp revealed 33 loci. Nine were found to be polymorphic including two phosphoglucose isomerase loci, a phosphoglucomutase locus, three esterase loci, a lactate dehydrogenase locus, a malate dehydrogenase locus, and transferrin. Five of these polymorphic loci are reported for the first time, the two phosphoglucose isomerase loci, a phosphoglucomutase locus and two of the three esterase loci. Differences in allele frequencies between the European and Chinese races were found at most loci.     

Enzyme polymorphisms in the European oyster, Ostrea edulis L.

Extracts of the digestive diverticula of more than 300 individuals from four geographically separated populations of the European oyster, Ostrea edulis L. have been examined by electrophoresis for esterase and phosphoglucose isomerase polymorphisms. Three regions of esterase variability have been detected in the electrophoretograms. It is inferred that the variants in two of these regions are governed by two co-dominant alleles at each of two loci. Two phenotypes of phosphoglucose isomerase, which is inferred to be a dimeric enzyme encoded by two alleles at a single locus, have been observed. Allele frequencies and phenotype distributions are compared in the four populations. It is concluded that the populations differ genetically at the loci investigated, the magnitude of the differences being generally greatest between different 'physiological races'.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion

Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.     

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.     

6-phosphogluconolactonase mutants of Escherichia coli and a maltose blue gene

Mutants lacking an enzyme of the oxidative branch of the hexose monophosphate shunt, 6-phosphogluconolactonase (pgl), have been selected as a new class of glucose-negative derivatives of a phosphoglucose isomerase (pgi) mutant. Glucose negativity is not as complete as in mutants lacking phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Pgi(+), pgl(-) strains have been constructed by transduction and grow almost normally on glucose. Genetic mapping shows that pgl lies between chlD and att-lambda, in the same position as and identical with a blu gene described by Adhya and Schwartz. These blu mutants grown on maltose were recognized by their property to turn blue after treatment with iodine. It is not known how phosphogluconolactonase deficiency causes this reaction; it might be related to accumulation of 6-phosphogluconolactone.     

Relationship between deficiency of phosphoglucose isomerase in Coprinus macrorhizus and fruiting body formation.

A mutant (pgi) of Coprinus macrorhizus deficient in phosphoglucose isomerase did not grow on fructose and grew poorly on glucose. The pgi mutation inhibited the formation of monokaryotic and dikaryotic fruiting bodies.     

Compartmentation in the induction of the hexose-6-phosphate transport system of Escherichia coli

The induction of the hexose-6-phosphate transport system was investigated. Glucose-6-phosphate (G6P) at concentrations as low as 10(-4)m was able to induce this system in wild-type cells, as well as in mutants lacking phosphoglucose isomerase or G6P dehydrogenase. Growth in the presence of fructose-6-phosphate (F6P) induced the system only if the cells contained phosphoglucose isomerase. Furthermore, glucose and F6P were found to induce the system only if the extracellular concentration of G6P became appreciable in the medium as a consequence of the leakage of intracellular G6P formed from the glucose or F6P. Intracellular G6P was not an inducer even at high concentrations. The metabolism of glucose inhibited the induction of the hexose-6-phosphate transport system. Hypotheses for this compartmentalization of inducer and membrane-associated induction are presented.     

Phosphoglucose isomerase in developing teeth

Summary The distribution of phosphoglucose isomerase in developing teeth of rats, using an indirect tetrazolium technique, is described. The demonstration of this enzyme, which occupies a position of considerable importance in carbohydrate metabolism, provides additional information regarding the biochemistry of odontogenic cells.     

Molecular characterization of pig muscle phosphoglucose isomerase

As a corollary to X-ray crystallographic work performed by H. Muirhead, detailed studies on crystalline pig muscle phosphoglucose isomerase have been conducted to establish its basic physical and chemical properties. The enzyme species being investigated by X-ray diffraction has been determined to be isoenzyme III. Its molecular weight in the native state was found to be 132,000, its s⁰₂₀,w value to be 7·25 S. The enzyme is composed of two subunits of equal molecular weight (66,000). Its amino acid composition is largely similar to that of rabbit muscle phosphoglucose isomerase, with the significant exception that the pig muscle isomerase contains only three sulfhydryl groups per polypeptide chain (two of them accessible to titration with p-mercuribenzoate) as compared with twice that number for the rabbit muscle enzyme. This low number of sulfhydryl groups is interpreted as being responsible for the ease with which heavy-atom, isomorphous derivatives could be prepared for the pig muscle enzyme by Shaw & Muirhead (1977).