The effect of substitution at C-2 of d-glucose 6-phosphate on the rate of dehydrogenation by glucose 6-phosphate dehydrogenase (from yeast and from rat liver)

1. The deoxyfluoro-d-glucopyranose 6-phosphates are substrates for both yeast and rat liver glucose 6-phosphate dehydrogenase. 2. The V(max.) values (relative to d-glucose 6-phosphate) were determined for a series of d-glucose 6-phosphate derivatives substituted at C-2. The V(max.) values decreased with increasing electronegativity of the C-2 substituent. This is consistent with a mechanism involving hydride-ion transfer. 3. 2-Deoxy-d-arabino-hexose 6-phosphate (2-deoxy-d-glucose 6-phosphate) showed substrate inhibition with the yeast enzyme but not with the rat liver enzyme. 4. 2-Amino-2-deoxy-d-glucose 6-phosphate (d-glucosamine 6-phosphate) was a substrate for the yeast enzyme but a competitive inhibitor for the rat liver enzyme. 5. Lineweaver-Burk plots for the d-glucose 6-phosphate derivatives with yeast glucose 6-phosphate dehydrogenase were biphasic.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase

An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.     

Inactivation and degradation of yeast glucose-6-phosphate dehydrogenase selectively modified by pyridoxal-5-phosphate

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.     

Dehydrogenation by gluconate 6-phosphate dehydrogenase of an isosteric phosphonate substrate analogue.

6,7-Dideoxy-D-gluco-heptonic-7-phosphonic acid, the isosteric phosphonate analogue of gluconate 6-phosphate, was prepared by incubation of the corresponding analogue of glucose 6-phosphate with glucose 6-phosphate dehydrogenase and NADP+ in the presence of an enzyme NADPH-NADP+ recycling system. The analogue of gluconate 6-phosphate is a substrate for yeast gluconate 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH 7.5 and 8.0. At both pH values the Km values are approx. 3-fold higher and the Vmax. values approx. 7-fold lower than those of the natural substrate.     

Eukaryotic glucose-6-phosphate dehydrogenases: Structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.     

Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae.

To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.     

Enhanced proteolysis of glucose-6-phosphate dehydrogenase in the presence of palmitoyl coenzyme A

Palmitoyl coenzyme A at concentrations below its critical micelle concentration increases the rate of proteolysis of baker's yeast glucose-6-phosphate dehydrogenase by proteinase A in the pH range 4-5. both glucose-6-phosphate and NADP protect glucose-6-phosphate dehydrogenase against proteolysis, but these protective effects are diminished in the presence of palmitoyl coenzyme A. Since palmitoyl coenzyme A is known to dissociate glucose-6-phosphate dehydrogenase into dimers, the results imply that the in vivo half life of glucose-6-phosphate dehydrogenase may be controlled by a process based on the regulation of the oligomeric structure of the enzyme by the collective actions of various molecules, including palmitoyl coenzyme A.     

The structural subunit of glucose-6-phosphate dehydrogenase (baker's yeast).

The subunit molecular weight of glucose-6-phosphate dehydrogenase (G6PD) from baker's yeast has been evaluated. The subunit molecular weight value is shown to be 25,500 daltons by analytical ultracentrifugation, SDS-polyacrylamide gel electrophoresis, and the number of peptides produced by CNBr cleavage. The number of NADP binding sites was determined to be one per 25,500 dalton unit.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Structural and kinetic differences between human and Aspergillus fumigatus D-glucosamine-6-phosphate N-acetyltransferase

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.     

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.     

Effects of AMP and Cibacron blue F3G-A on the fructose 6-phosphate binding of yeast phosphofructokinase.

The positive effector 5′-AMP of yeast phosphofructokinase does not influence the binding of fructose 6-phosphate to the enzyme. Cibacron blue F3G-A considered an ATP analogue decreases the affinity of the enzyme to fructose 6-phosphate without exerting an effect on the cooperativity of fructose 6-phosphate binding. The peculiarities of the interactions of AMP and Cibacron blue with fructose 6-phosphate binding demonstrate compatibility of the allosteric kinetics with the binding behavior of the enzyme.     

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.     

Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes

Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower K(m) values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 degrees C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses.     

Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor

The interactions of the bovine cation-dependent mannose 6-phosphate receptor with monovalent and divalent ligands have been studied by equilibrium dialysis. This receptor appears to be a homodimer or a tetramer. Each mole of receptor monomer bound 1.2 mol of the monovalent ligands, mannose 6-phosphate and pentamannose phosphate with Kd values of 8 X 10(-6) M and 6 X 10(-6) M, respectively and 0.5 mol of the divalent ligand, a high mannose oligosaccharide with two phosphomonoesters, with a Kd of 2 X 10(-7) M. When Mn2+ was replaced by EDTA in the dialysis buffer, the Kd for pentamannose phosphate was 2.5 X 10(-5) M. By measuring the affinity of the cation-dependent and cation-independent mannose 6-phosphate receptors for a variety of mannose 6-phosphate analogs, we conclude that the 6-phosphate and the 2-hydroxyl of mannose 6-phosphate each contribute approximately 4-5 kcal/mol of Gibb's free energy to the binding reaction. Neither receptor appears to interact substantially with the anomeric oxygen of mannose 6-phosphate. The receptors differ in that the cation-dependent receptor displays no detectable affinity for N-acetylglucosamine 1'-(alpha-D-methylmannopyranose 6-monophosphate) whereas this ligand binds to the cation-independent receptor with a poor, but readily measurable Kd of about 0.1 mM. The spacing of the mannose 6-phosphate-binding sites relative to each other may also differ for the two receptors.     

A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases

The soluble hydrolases of the mammalian lysosome are marked for delivery to this organelle by the addition of mannose 6-phosphate to their N-glycans. Two related mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi network (TGN) and deliver the hydrolases to the late endosome. In contrast, the vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain 6-phosphate monoesters on their N-glycans, and the only sorting receptor so far identified in this organism is the product of the VPS10 gene. This protein also cycles between the Golgi and the late endosome, but is unrelated to the vertebrate MPRs, and recognizes a specific amino acid sequence of carboxypeptidase Y (CPY). This has led to the notion that although yeast and mammals share many components in Golgi to endosome traffic, they use unrelated receptor systems to sort their abundant soluble hydrolases. In this paper, we report that the yeast genome does in fact contain an uncharacterized ORF (YPR079w) that encodes a membrane protein that is distantly related to mammalian MPRs. The protein encoded by this gene (which we term MRL1) cycles through the late endosome. Moreover, there is a strong synergistic effect on the maturation of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases.     

Free radical inactivation of glucose-6-phosphate dehydrogenase in Saccharomyces cerevisiae in vitro

Partial purification and in vitro inactivation of glucose-6-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae in the Fe2+/H2O2 oxidation system were conducted. At the protein concentration 1.5 mg/ml, the enzyme lost 50% of activity within 5 minutes of incubation in presence of 2 mM hydrogen peroxide and 3 mM ferrous sulphate. The inactivation extent depended on time and concentrations of FeSO4 and H2O2. EDTA, ADP and ATP at concentration 0.5 mM enhanced inactivation. At the same time, the presence of 0.5 mM NADPH, 1 mM glucose-6-phosphate, 10 mM mannitol, 30 mM dimethylsulphoxide or 20 mM urea diminished this process. In comparison with native enzyme, index S(0,5) of the partially inactivated enzyme for glucose-6-phosphate was 2.1-fold higher, but for NADP it was 1,6-fold lower. Maximal activity of the partially inactivated enzyme was 3-5-fold lower than that of native one.     

Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex

Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.