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PubMed1994

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.

Acrylamides↗
PubMed1995

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.

Adenosine Triphosphate↗
PubMed1997

Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases.

Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff-base intermediate between dihydroxyacetone and the epsilon-amino group of a lysine residue at the active site of the enzyme is formed. This Schiff-base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 A resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.

Catalysis↗
PubMed2007

Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway.

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.

Adenine Nucleotides↗
PubMed1992

Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose-limited continuous culture.

In this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose-limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g-1 at t = 0 to 25 nmol g-1 at t = 1.5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.

Adenosine Triphosphate↗
PubMed1993

Phosphofructokinase from liver of the rainbow trout, Oncorhynchus mykiss.

Phosphofructokinase (PFK) from liver of the rainbow trout Oncorhynchus mykiss was purified to homogeneity with a recovery of 35% of total activity. The purified enzyme was a homotetramer with a native molecular weight of 297,000 +/- 16,000 and a subunit M(r) of 76,000 +/- 3000. Arrhenius plots of enzyme activity were linear over 5-27 degrees C with an activation energy of 52.3 +/- 2.1 kJ/mol. The binding of fructose 6-phosphate was cooperative. High ATP increased the Hill coefficient and produced a marked allotropic inhibition of the enzyme activity. The affinity of the enzyme for fructose 6-phosphate was increased by the addition of the enzyme activators such as inorganic phosphate, ammonium ions, AMP, and fructose 2,6-bisphosphate; the activators also reduced the inhibitory effect of ATP. Trout liver PFK was activated by phosphoenolpyruvate at physiological concentrations but was not affected by citrate.

Adenosine Monophosphate↗
PubMed1995

Metabolism of fructose-3-phosphate in the diabetic rat lens.

Fructose-3-phosphate and sorbitol-3-phosphate are produced in diabetic rat lenses by a 3-phosphokinase. While sorbitol-3-phosphate appears to be an inert polyol phosphate, fructose-3-phosphate is a potent cross-linking agent and a potential in vivo source of 3-deoxyglucosone. The objective of this study was to investigate the production and decomposition of fructose-3-phosphate in the diabetic rat lens. The results indicate that this metabolite achieves a steady-state concentration of almost 1 mumol/g wet weight within 2 weeks after the onset of diabetes. These steady-state levels appear to be a consequence of a balance between its production from fructose and its further decomposition to 3-deoxyglucosone. This conclusion is supported by results from disappearance of fructose-3-phosphate in insulin-treated diabetic rats and in vitro incubations of fructose-3-phosphate with amines where production of 3-deoxyglucosone was detected using a number of different methods including mass spectrometry. In agreement with these results, elevated concentrations of 3-deoxyglucosone along with its detoxification product, 3-deoxyfructose, were detected in the diabetic rat lenses. Other sugars and sugar phosphates which were detectable in the diabetic rat lenses were glucose, fructose, glucose-6-phosphate, fructose-6-phosphate, and sedoheptulose-7-phosphate. In conclusion, results from this study suggest that fructose-3-phosphate and 3-deoxyglucosone are likely to be important contributors to the process of nonenzymatic glycation in diabetic rat lenses.

Animals↗
PubMed1996

Allosteric regulation of liver phosphorylase a: revisited under approximated physiological conditions.

Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase a and b. The a form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by approximately 10- and 20-fold. Thus, unless phosphorylase a is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase a activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase a was used. Activity was measured in the direction of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rate conditions. Both a Km and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the Km for Pi by approximately 50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase a activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase a activity or the regulation of phosphorylase a activity.

Adenine Nucleotides↗
PubMed1999

Changes in fructose-induced production of glucose in the rat liver following partial hepatectomy.

The fructose-induced production of glucose in the liver after partial hepatectomy (PH) was evaluated by using the liver-perfusion system. There was no significant difference in plasma glucose level between hepatectomized (HX) and sham-operated (SO) rats at 24 h after surgery, and, thereafter, almost similar levels were obtained in both groups. However, the level of serum free fatty acids (FFA) was significantly higher in HX rats than that in SO rats at 24 and 48 h after surgery. When both groups of rats were given fructose by gavage, the increment of plasma glucose was significantly larger in HX rats than in SO rats. Lactate infusion failed to increase the rate of glucose production in perfused livers of both HX and SO rats and there was no significant difference in the activity of hepatic phosphoenolpyruvate carboxykinase. By contrast, fructose infusion elicited a large increase in glucose production in the perfused livers of HX rats at 24 and 48 h after PH. The increase was closely associated with not the change in fructose 2,6-bisphosphate levels but the increment of the intracellular levels of citrate. Treatment of octanoate or oleate, which supplies acetyl-CoA via fatty acid oxidation, mimicked the fructose-induced increase in glucose production in SO rats with a concomitant increase in hepatic levels of citrate. These results suggest that the oxidation of FFA may play an important role in glucose production induced by fructose administration during the early phase of liver regeneration.

Acetyl Coenzyme A↗
PubMed2000

Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties.

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.

Acrylamide↗
PubMed2000

Site-directed mutagenesis of the fructose 6-phosphate binding site of the pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica.

Attempts to define the active site of pyrophosphate-dependent phosphofructokinase (PPi-PFK) using homology modeling based on the three-dimensional structure of the ATP-dependent PFKs from bacteria have been frustrated by low sequence identity between PPi- and ATP-PFKs in their carboxyl terminal halves. In the current study, alanine scanning mutagenesis of residues in the carboxyl terminal half of the PPi-PFK of Entamoeba histolytica coupled with comparative sequence analysis and computational modeling is used to identify residues that contribute to fructose 6-phosphate (fructose 6-P) binding. Of seven alanine mutants that were generated by site-directed mutagenesis, Arg377, Ser392, Arg405, Lys408, His415, His416, and Arg423, only the last mutant, Arg423Ala, was found to have dramatically lower affinity for fructose 6-P. Mutation of Arg 423 decreased k(cat) by 10,000-fold and decreased apparent affinity for fructose 6-P by 126-fold, while the K(m) for PPi increased only 4-fold. The second greatest effect was seen with Arg377Ala, which had a nearly 10-fold decrease in apparent affinity and an approximate 60-fold decrease in maximal activity. Another residue, Tyr420, was chosen for mutagenesis by its complete identity in all other PPi-PFK. This residue and its homologue in Escherichia coli ATP-PFK, His249, were mutated and shown to be very important for substrate binding in both enzymes.

Alanine↗
PubMed2001

Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Adenosine Triphosphate↗
PubMed2002

A radiometric assay for glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a method for the synthesis and purification of the substrate, fructose 6-phosphate, and methods for a radiometric assay of human GFAT1 that can be performed in either of two formats: a small disposable-column format and a high-throughput 96-well-plate format. The method performed in the column format can detect 1 pmol of glucosamine 6-phosphate, much less than that required by previously published assays that measure GlcN 6-phosphate. The column assay demonstrates a broad linear range with low variability. In both formats, the assay is linear with time and enzyme concentration and is highly reproducible. This method greatly improves the sensitivity and speed with which GFAT1 activity can be measured and facilitates direct kinetic measurement of the transferase activity.

Animals↗
PubMed1996

Isozyme analysis of human polymorphonuclear leukocyte phosphofructokinase from insulin resistant individuals.

Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies and column chromatography on QAE-Sephadex in three different groups: control, type II diabetic, and obese individuals. It was found that PMN phosphofructokinase in the three groups consists mainly of a mixture of L4 and M4 homotetramers with possibly some hybrid forms. The predominant subunit was the L-type. A 24% decrease in the specific activity of the L-type isozyme was observed and an intermediate form (I-isozyme) having 23% of the total activity in diabetic individuals appeared. In obese individuals a 30% decrease was observed in the activity of M-type isozyme and 9% of the total activity corresponded to the intermediate form. Kinetic studies showed different regulatory properties among the isozymes from the three groups. The lower PFK activity found in diabetic and obese individuals can be associated with the decreased activity in the L-type isozyme (for diabetic individuals) and in the M-type isozyme (for obese individuals); the lower activity can also be associated with the four times lower affinity for F-6-P showed by the M-type isozyme, the decreased sensitivity to ATP inhibition (for both isozymes), and the appearance of an intermediate form with a different kinetic behaviour.

Adenosine Triphosphate↗
PubMed2000

ADP modulates the dynamic behavior of the glycolytic pathway of Escherichia coli.

A mathematical model that includes biochemical interactions among the PTS system, phosphofructokinase (PFK), and pyruvate kinase (PK) is used to evaluate the dynamic behavior of the glycolytic pathway of Escherichia coli under steady-state conditions. The influence of ADP, phosphoenolpyruvate (PEP), and fructose-6-phosphate (F6P) on the dynamic regulation of this pathway is also analyzed. The model shows that the dynamic behavior of the system is affected significantly depending on whether ADP, PEP, or F6P is considered constant a steady state. Sustained oscillations are observed only when dADP/dt not equal 0 and completely suppressed if dADP/dt = 0 at any steady-state value. However, when PEP or F6P is constant, the system evolves toward the formation of stable limit cycles with periods ranging from 0.2 min to hours.

Adenosine Diphosphate↗
PubMed2002

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.

Adenosine Triphosphate↗
PubMed1993

In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium.

Evidence has been presented suggesting that the fructose repressor, FruR, is a pleiotropic transcriptional regulatory protein controlling the expression of numerous operons concerned with carbon metabolism in Escherichia coli and Salmonella typhimurium. We have conducted in vitro DNA binding studies to ascertain the nature of the DNA sequences to which FruR binds. Employing both DNA band migration retardation and DNase I footprint analyses, FruR was found to bind to two operators within the regulatory region preceding the structural genes of the fructose operon, fruB(MH)KA. These two operators, O1 and O2, comprise nearly identical palindromes of 12 bp with a half-site of TGAAAC. The binding of FruR to these inverted repeats was found to be reversed by inclusion of micromolar concentrations of fructose-1-phosphate. The two operators are located between the single putative promoter of the fructose operon and the translational initiation site of the fruB gene. Other regulated operons were shown to bind FruR to a single site upstream of the first structural gene as follows: (1) ppsA (positive regulation); (2) icd (positive regulation); (3) aceB (positive regulation); and (4) pts (negative regulation). In all cases, low concentrations of fructose-1-phosphate displaced the protein from the DNA. The binding sites were determined, and a FruR consensus sequence was established. Computer searches revealed the presence of this sequence in numerous functionally diverse operons, implying that FruR is a global transcriptional regulatory protein in enteric bacteria.

Allosteric Regulation↗