Characterization of an enzyme which catalyzes isomerization and epimerization of D-erythrose 4-phosphate

The enzyme which catalyzes the conversion of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G-100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43 000. From the gas chromatography/mas spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4-phosphate was present as D-erythrulose 4-phosphate after equilibration. The purified enzyme, which is tentatively called 'erythrose-4-phosphate isomerase' had no significant isomerase activities on D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-glucose 6-phosphate and D-fructose 6-phosphate, but a strong D-ribulose-5-phosphate 3-epimerase activity was co-purified with the erythrose-4-phosphate isomerase activity through every step in the isolation. Both the erythrose-4-phosphate isomerase and D-ribulose-5-phosphate 3-epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likely, that both activities are catalyzed by the single enzyme protein.     

Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-d-ribose-5-phosphate aldolase

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.     

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.     

A novel phosphoglucose isomerase (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A resolution

The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate.     

Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate

In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.     

Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.Abbreviations RPI ribose-5-phosphate isomerase - OPPP oxidative pentose phosphate pathway - CNBr cyanogen bromide - R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate     

Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: Purification and molecular analysis of the enzyme

Purification and molecular analysis of ribose-5-phosphate isomerase (EC5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzymewas enriched from a haploid deletion mutant containing the wild-type gene ona multicopy plasmid elaborating the following steps: ammonium sulphateprecipitation, interfacial salting out on Sepharose 6B, high performanceliquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. Theenzyme activity was found to be rather unstable possibly caused by removalof stabilizing cofactors or proteins during the purification procedure.The purified enzyme showed a hyperbolic dependence on the substrateribose-5-phosphate with a K_m-value of 1.6±0.3 mmol/l.For the native enzyme a molecular mass of 115±10 kDa was determinedas found by saccharose density gradient centrifugation, sedimentationequilibrium analysis, size exclusion chromatography and polyacrylamide gelelectrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresisand Western blotting revealed one band with a molecular mass of 31±2kDa. Thus, the native enzyme is composed of four subunits of identicalsize.The molecular mass of the subunit and the identified N-terminal sequenceof 33 amino acids fits well the 258 amino acid protein encoded by the S.cerevisiae RKI open reading frame, which was characterized previously onlyby increasing specific activities of ribose-5-phosphate isomerase in cellsafter cloning the gene. On the basis of the conserved amino acids analignment of the amino acid sequence of ribose-5-phosphate isomerase fromyeast with those of the enzyme from mouse, spinach and Escherichia coli ispresented.     

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by ¹³C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically ¹³C-labeled at C1 and C3, formed acetate and small amounts of lactate. The ¹³C-labeling patterns, analyzed by ¹H- and ¹³C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.Abbreviations. KDG 2-Keto-3-deoxygluconate - KDPG 2-Keto-3-deoxy-6-phosphogluconate - FBP Fructose-1,6-bisphosphate - TIM Triosephosphate isomerase - GAP Glyceraldehyde-3-phosphate - PEP Phosphoenolpyruvate - PTS Phosphotransferase - 1-PFK Fructose 1-phosphate kinase An erratum to this article can be found at     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.     

Regulation of resynthesis of the CO2-acceptor in photosynthesis. Feedback inhibition of transketolase

The presence of ribulose-5-phosphate epimerase (EC 5.1.3.1, epimerase) in samples of ribose-5-phosphate isomerase (EC 5.3.1.6, isomerase) obtained from spinach ( Spinacea aleracea L. cv. Bloomsdale Long Standing) was determined using (i) a sampling procedure which measured the quantity of xylulose-5-phosphate formed in the reaction mixture and (ii) a coupled enzyme assay in which the rate of oxidation of NADH was measured after establishing steady-state concentrations of xylulose-5-phosphate, dihydroxacetonephosphate and glyceraldehyde-3-phosphate by the action of epimerase, transketolase (EC 2.2.1.1), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). In preparations where the ratio of isomerase to epimerase activities was less than 100, both assay procedures yielded valid indications of epimerase activity. The steady-state assay system was found, however, to seriously underestimate epimerase activity in enzyme preparations which were enriched in isomerase. Cross plots of epimerase activity determined by the sampling and steady-state procedures demonstrated that an inhibitor of the coupling enzyme mixture was formed in the presence of high relative concentrations of the isomerase. The inhibited coupling enzyme mixture was fully active with glycer-aldehyde-3-phosphate. Inhibition of the coupling enzyme mixture was attributed to transketolase. Feedback inhibition of transketolase is proposed to be of physiological significance in the photosynthesis cycle, operating to restrict resynthesis of CO₂-acceptor under conditions where high steady-state concentrations of the intermediates of the photosynthesis cycle are maintained.     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings.The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.     

Identification of free mannose and partial characterization of a mannose-6-phosphate isomerase from Lilium longiflorum bulbs

The existence of free mannose in storage bulbs of Lilium longiflorum Thunb, was established using preparative high performance liquid chromatography, gas chromatography and gas chromatography-mass spectroscopy. Free mannose was not detected in developing (importing) bulb tissues. Mannose, a relatively rare hexose in plant tissue, probably arises from the hydrolysis of glucomannan, a hemicellulosic carbohydrate polymer known to be present in Lilium storage tissues. A calculation of total mannose residues per bulb (prior to versus after reserve hydrolysis and export) indicated that mannose is metabolized, probably in sucrose biosynthesis. A mannose-6-phosphate isomerase (EC 5.3.1.8) was isolated from Lilium bulbs and purified 155-fold with 29% yield. The molecular weight of the enzyme was estimated by gel filtration to be 64 kDa, and the K_m for mannose-6-phosphate was 0.42 m M . It is concluded that glucomannan is functioning as a reserve carbohydrate in Lilium storage tissues and that the mannose-6-phosphate isomerase is responsible for the entry of mannose into the sucrose biosynthetic pathway.     

Structural basis for phosphomannose isomerase activity in phosphoglucose isomerase from Pyrobaculum aerophilum: a subtle difference between distantly related enzymes

The crystal structure of a dual-specificity phosphoglucose/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum (PaPGI/PMI) has been determined in complex with glucose 6-phosphate at 1.16 A resolution and with fructose 6-phosphate at 1.5 A resolution. Subsequent modeling of mannose 6-phosphate (M6P) into the active site of the enzyme shows that the PMI activity of this enzyme may be due to the additional space imparted by a threonine. In PGIs from bacterial and eukaryotic sources, which cannot use M6P as a substrate, the equivalent residue is a glutamine. The increased space may permit rotation of the C2-C3 bond in M6P to facilitate abstraction of a proton from C2 by Glu203 and, after a further C2-C3 rotation of the resulting cis-enediolate, re-donation of a proton to C1 by the same residue. A proline residue (in place of a glycine in PGI) may also promote PMI activity by positioning the C1-O1 region of M6P. Thus, the PMI reaction in PaPGI/PMI probably uses a cis-enediol mechanism of catalysis, and this activity appears to arise from a subtle difference in the architecture of the enzyme, compared to bacterial and eukaryotic PGIs.     

Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12),a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of Camelusdromedarius.The protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere investigated.The native protein is a homotetramer with an apparent molecular weight of approximately146 kDa.Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of 7.2.The optimum pH of the purified enzyme was 7.8.Studies on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/mol.The apparent K_m values for NAD~ and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The V_(max) of the purified protein was estimated to be 52.7±5.9 U/mg.These kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.     

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate

Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0muM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40muM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.     

Glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.