Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

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.     

The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate

Phosphoglucose isomerase (PGI) catalyzes the isomerization of D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis. Analysis of previously reported X-ray crystal structures of PGI without ligand, with the cyclic form of F6P, or with inhibitors that mimic the cis-enediol intermediate led to proposed mechanisms for the ring opening and isomerization steps in the multistep catalytic mechanism. To help complete our model of the overall mechanism, information is needed about the state of PGI between the ring opening and isomerization steps, in other words, a structure of the enzyme complexed with the open form of a substrate or an analog. Here, we report the crystal structure of rabbit PGI complexed with D-sorbitol-6-phosphate (S6P), an analog of the open chain form of G6P, at 2.0 A resolution. As was seen in the PGI/F6P structure, a helix containing amino acid residues 512-520 is found in the "out" position, which provides sufficient space in the active site for a substrate in its cyclic form and which is probably the location of that helix just after ring opening (or just before ring closure). However, the S6P ligand is in an extended conformation, as was seen previously with ligands that mimic the cis-enediol intermediate. The extended conformation enables the ligand to interact with Glu357, which transfers a proton during the isomerization step. The PGI/S6P structure represents the conformation of the enzyme and substrate between the ring opening (or ring closing) step and the isomerization step and helps to complete the model for PGI's catalytic mechanism.     

Structural evidence for a hydride transfer mechanism of catalysis in phosphoglucose isomerase from Pyrococcus furiosus

In the Euryarchaeota species Pyrococcus furiosus and Thermococcus litoralis, phosphoglucose isomerase (PGI) activity is catalyzed by an enzyme unrelated to the well known family of PGI enzymes found in prokaryotes, eukaryotes, and some archaea. We have determined the crystal structure of PGI from Pyrococcus furiosus in native form and in complex with two active site ligands, 5-phosphoarabinonate and gluconate 6-phosphate. In these structures, the metal ion, which in vivo is presumed to be Fe2+, is located in the core of the cupin fold and is immediately adjacent to the C1-C2 region of the ligands, suggesting that Fe2+ is involved in catalysis rather than serving a structural role. The active site contains a glutamate residue that contacts the substrate, but, because it is also coordinated to the metal ion, it is highly unlikely to mediate proton transfer in a cis-enediol mechanism. Consequently, we propose a hydride shift mechanism of catalysis. In this mechanism, Fe2+ is responsible for proton transfer between O1 and O2, and the hydride shift between C1 and C2 is favored by a markedly hydrophobic environment in the active site. The absence of any obvious enzymatic machinery for catalyzing ring opening of the sugar substrates suggests that pyrococcal PGI has a preference for straight chain substrates and that metabolism in extreme thermophiles may use sugars in both ring and straight chain forms.     

Localization of Phosphoglucose Isomerase in Escherichia coli and Its Relation to the Induction of the Hexose Phosphate Transport System

The localization of phosphoglucose isomerase (PGI) was studied in relation to the induction of hexose phosphate uptake in Escherichia coli. The uptake system is induced only by extracellular glucose-6-phosphate (G6P); there is no induction by intracellular G6P. Fructose-6-phosphate (F6P) is an indirect inducer, and isomerization of F6P to G6P must occur before induction. PGI has been considered to be an internal enzyme; therefore, uptake of F6P by noninduced cells and leakage of the G6P formed would be required for induction. In this study, it was concluded that part of the PGI activity is located in the cell surface because: (i) uninduced, intact cells are able to convert F6P to G6P, whereas the activity of G6P dehydrogenase is not detectable; (ii) when cells are subjected to osmotic shock, about 10% of the PGI activity is found in the shock fluid; and (iii) sorbitol-6-phosphate (S6P) inhibits both PGI activity of whole cells and the induction of hexose phosphate transport system by F6P. S6P was not taken by intact cells. The data indicate that the isomerization of F6P to G6P can take place on the cell surface, and this explains the indirect induction of hexose phosphate transport by F6P.     

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.     

Crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-D-arabinonate identifies the role of Glu357 in catalysis

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the second step in glycolysis, the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. We determined the X-ray crystal structure of rabbit PGI complexed with a competitive inhibitor of isomerase activity, 5-phospho-D-arabinonate (5PAA), at 1.9 A resolution. 5PAA is a better mimic of the proposed cis-enediol(ate) intermediate than 6-phospho-D-gluconate, which was used in a previously reported crystal structure of rabbit PGI. The orientation of 5PAA bound in the enzyme active site predicts that active site residue Glu357 is the residue that transfers a proton between C2 and C1 of the proposed cis-enediol(ate) intermediate. Amino acid residues Arg272 and Lys210 are predicted to be involved in stabilizing the negative charge of the intermediate.     

Glucose 6-Phosphate dehydrogenase variants: A unique variant (G6PD Kobe) showed an extremely increased affinity for galactose 6-phosphate and a new variant (G6PD Sapporo) resembling G6PD Pea Ridge

Summary Two new glucose 6-phosphate dehydrogenase (G6PD) variants associated with chronic nonspherocytic hemolytic anemia were discovered. G6PD Kobe was found in a 16-year-old male associated with hemolytic crisis after upper respiratory infection. The enzyme activity of the variant was about 22% of that of the normal enzyme. The main enzymatic characteristics were slower than normal anodal electrophoretic mobility, high Km G6P, increased thermal-instability, an acidic pH optimum, and an extremely increased affinity for the substrate analogue, galactose 6-phosphate (Gal-6P).G6PD Sapporo was found in a 3-year-old male associated with drug-induced hemolysis. The enzyme activity was extremely low, being 3.6% of normal. In addition, this variant showed high Ki NADPH and thermal-instability.G6PD Kobe utilized the artificial substrate Gal-6P effectively as compared with the common natural substrate, glucose 6-phosphate. In G6PD Sapporo, NADPH could not exert the effect of product inhibition. The structural changes of these variants are expected to occur at the portions inducing conformational changes of the substrate binding site of the enzyme.     

Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate

Transketolase is a prominent thiamin diphosphate-dependent enzyme in sugar metabolism that catalyzes the reversible transfer of a 2-carbon dihydroxyethyl fragment between a donor ketose and an acceptor aldose. The X-ray structures of transketolase from E. coli in a covalent complex with donor ketoses d-xylulose 5-phosphate (X5P) and d-fructose 6-phosphate (F6P) at 1.47 A and 1.65 A resolution reveal significant strain in the tetrahedral cofactor-sugar adducts with a 25-30 degrees out-of-plane distortion of the C2-Calpha bond connecting the substrates' carbonyl with the C2 of the cofactor's thiazolium part. Both intermediates adopt very similar extended conformations in the active site with a perpendicular orientation of the scissile C2-C3 sugar bond relative to the thiazolium ring. The sugar-derived hydroxyl groups of the intermediates form conserved hydrogen bonds with one Asp side chain, with a cluster of His residues and with the N4' of the aminopyrimidine ring of the cofactor. The phosphate moiety is held in place by electrostatic and hydrogen-bonding interactions with Arg, His, and Ser side chains. With the exception of the thiazolium part of the cofactor, no structural changes are observable during intermediate formation indicating that the active site is poised for catalysis. DFT calculations on both X5P-thiamin and X5P-thiazolium models demonstrate that an out-of-plane distortion of the C2-Calpha bond is energetically more favorable than a coplanar bond. The X-ray structure with the acceptor aldose d-ribose 5-phosphate (R5P) noncovalently bound in the active site suggests that the sugar is present in multiple forms: in a strained ring-closed beta-d-furanose form in C2-exo conformation as well as in an extended acyclic aldehyde form, with the reactive C1 aldo function held close to Calpha of the presumably planar carbanion/enamine intermediate. The latter form of R5P may be viewed as a near attack conformation. The R5P binding site overlaps with those of the leaving group moieties of the covalent donor-cofactor adducts, demonstrating that R5P directly competes with the donor-derived products glyceraldehyde 3-phosphate and erythrose 4-phosphate, which are substrates of the reverse reaction, for the same docking site at the active site and reaction with the DHEThDP enamine.     

The reaction mechanism of type I phosphomannose isomerases: new information from inhibition and polarizable molecular mechanics studies

Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI. Its K(i) value of 41 nM for human PMI is the lowest value ever reported for an inhibitor of PMI. 5-Phospho-D-arabinonhydrazide, a neutral analogue of the reaction intermediate 1,2-cis-enediol, is about 15 times less efficient at inhibiting both enzymes, in accord with the anionic nature of the postulated high-energy reaction intermediate. Using the polarizable molecular mechanics, sum of interactions between fragments ab initio computed (SIBFA) procedure, computed structures of the complexes between Candida albicans (Type I) PMI and the cyclic substrate β-D-mannopyranose 6-phosphate (β-M6P) and between the enzyme and the high-energy intermediate analogue inhibitor 5PAH are reported. Their analysis allows us to identify clearly the nature of each individual active site amino acid and to formulate a hypothesis for the overall mechanism of the reaction catalyzed by Type I PMIs, that is, the ring-opening and isomerization steps, respectively. Following enzyme-catalyzed ring-opening of β-M6P by zinc-coordinated water and Gln111 ligands, Lys136 is identified as the probable catalytic base involved in proton transfer between the two carbon atoms C1 and C2 of the substrate D-mannose 6-phosphate.     

Transmembrane segment 11 of UhpT, the sugar phosphate carrier of Escherichia coli, is an alpha-helix that carries determinants of substrate selectivity

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P(i)-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys(391), a member of an intrahelical salt bridge (Asp(388)/Lys(391)) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an alpha-helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.     

Importance of product/reactant equilibration in the kinetics of the phosphoglucose isomerization reaction by differential stopped flow microcalorimetry

The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P --> G6P) by baker's yeast phosphoglucose isomerase (PGI) with regard to k(cat) and K(m) were determined from analysis of differential stopped flow microcalorimeter measurements using the integrated form of the Michaelis-Menten rate equation. Values for K(m) (F6P --> G6P) that were determined at pH 8.0 and ionic strength 0.1M at 293.4, 298.4, 303.4, and 311.5K exhibited a linear dependence on the substrate concentration at each temperature because of the substrate-product equilibrium. The minimum values for K(m) ranged from 2.62+/-0.55 mM at 293.4K to 7.8+/-4.8mM at 311.5K and were the same as the minimum values for the reverse reaction (G6P --> F6P) at 293.4 K and 298.4 K. Minimum values for k(cat) increased with temperature, from 2.78+/-0.34s(-1) at 293.4K to 11.4+/-1.0s(-1) at 311.5K, and for the reverse reaction, G6P --> F6P, from 0.852+/-0.086 s(-1) at 293.4K to 1.46+/-0.06s(-1) at 298.4K. The enzyme efficiency at 311.5K is close to the collision rate for a diffusion-controlled process in solution. The [F6P]/[G6P] equilibrium constants were determined from comparison of the values of k(cat) in both directions and were 0.307+/-0.053 at 293.4K and 0.395+/-0.033 at 298.4K. The heats of reaction in the F6P --> G6P direction increased from -8.96+/-0.26 kJmol(-1) at 311.5K to -8.27+/-0.40 kJmol(-1) at 293.4K, a value in fair agreement with 7.01+/-0.32 kJmol(-1) in the opposite G6P --> F6P direction.     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Mechanism-based inactivation of rabbit muscle phosphoglucomutase by nojirimycin 6-phosphate

Nojirimycin 6-phosphate (N6P) was tested as a substrate and inhibitor for phosphoglucomutase (PGM). In the absence of glucose 1,6-bisphosphate (GBP), the incubation of PGM and N6P resulted in the complete inactivation of all enzyme activity. When equimolar amounts of N6P and GBP were incubated together with PGM, the GBP was quantitatively converted to glucose 6-phosphate (G6P) and phosphate. At higher ratios of GBP and N6P (greater than 100) the final concentration of G6P produced was found to be 19 times the initial N6P concentration. These results have been interpreted to suggest that the phosphorylated form of PGM catalyzes the phosphorylation of N6P at C-1. This intermediate rapidly eliminates phosphate to form an imine and the dephosphorylated enzyme. The dephosphorylated enzyme is rapidly rephosphorylated by GBP and forms G6P. The imine is nonenzymatically hydrated back to N6P. Occasionally (5%) the imine isomerizes to a compound that is not processed by PGM.     

Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.     

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.     

Crystal structures of mouse autocrine motility factor in complex with carbohydrate phosphate inhibitors provide insight into structure-activity relationship of the inhibitors

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is identical to the extracellular cytokines neuroleukin and maturation factor and, interestingly, to the intracellular enzyme phosphoglucose isomerase. The cytokine activity of AMF is inhibited by carbohydrate phosphate compounds as they compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven transmembrane helix protein. Here, we report the first comprehensive high-resolution crystal structure analyses of the inhibitor-free form and the eight types of inhibitor (phosphate, erythrose 4-phosphate (E4P), arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P), 6-phosphogluconic acid (6PGA), fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), or mannose 6-phosphate (M6P)) complexes of mouse AMF (mAMF). We assayed the inhibitory activities of these inhibitors against the cytokine activity of mAMF. The inhibitory activities of the six-carbon sugars (G6P, F6P, M6P, and 6PGA) were found to be significantly higher than those of the four or five-carbon sugars (E4P or A5P). The inhibitory activities clearly depend on the length of the inhibitor molecules. A structural comparison revealed that a water-mediated hydrogen bond between one end of the inhibitor and a rigid portion of the protein surface in the shorter-chain inhibitor (E4P) complex is replaced by a direct hydrogen bond in the longer-chain inhibitor (6PGA) complex. Thus, to obtain a new compound with higher inhibitory activities against AMF, water molecules at the inhibitor binding site of AMF should be replaced by a functional group of inhibitors in order to introduce direct interactions with the protein surface. The present structure-activity relationship studies will be valuable not only for designing more effective AMF inhibitors but also for studying general protein-inhibitor interactions.