Metabolic control and the microenvironment.
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Animals↗
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Metabolic effects of fructose in the liver.
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AMP Deaminase↗
The fructose 6-phosphate/fructose 1, 6 bisphosphate cycle.
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Animals↗
Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5'-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis.
Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.
Adenosine Triphosphate↗
A high-throughput screening for phosphatases using specific substrates.
A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.
Alkaline Phosphatase↗
Examination of MgATP binding in a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus.
A tryptophan-shift variant of Bacillus stearothermophilus phosphofructokinase (BsPFK), W179F/F76W, was constructed to evaluate the binding and allosteric characteristics associated with MgATP. W179F/F76W BsPFK has a specific activity of 77+/-1 U/mg at pH 7 and 25 degrees C, which is a 35% decrease compared to the wild-type enzyme. The K(m) for MgATP increases from 43+/-3 microM for wild-type BsPFK to 160+/-7 microM in the variant. Binding and allosteric interaction between Fru-6-P and PEP for the variant are similar to those of the wild-type enzyme. W179F/F76W BsPFK has distinct fluorescence properties relative to wild-type or other tryptophan-shifted mutants of BsPFK. The binding of MgATP produces an 80% decrease in the fluorescence intensity while MgADP causes a 70% decrease. Capitalizing on these fluorescence changes, dissociation constants of 30+/-1 microM and 0.53+/-0.02 mM were measured for MgATP and MgADP, respectively. In addition, PEP was shown to enhance MgATP binding by 2.6-fold.
Adenosine Diphosphate↗
Carnosine protects against the inactivation of esterase induced by glycation and a steroid.
Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes. Esterase activity in the lens is decreased in senile cataract and diabetes. Previously, we reported that glycation and a steroid inactivate esterase. Here we tested the inactivation of esterase with fructose, fructose 6-phosphate (F6P) and ribose as model glycation reactions and prednisolone-21-hemisuccinate (P-21-H) as a model steroid and investigated the ability of carnosine to protect esterase against inactivation. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. The modified esterase was examined electrophoretically. The esterase was progressively inactivated by F6P, fructose, ribose and P-21-H. P-21-H was more effective than the sugars. Carnosine significantly inhibited the inactivation of esterase induced by all four compounds. Carnosine decreased the extent of the cross-linking. These results provide further evidence for carnosine's role as an anti-glycation compound. It is also proposed that carnosine may be an anti-steroid agent.
Animals↗
Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction.
The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.
Adaptor Proteins, Signal Transducing↗
Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle.
The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.
Adenosine Triphosphate↗
Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis.
Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K(m) of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K(i) was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 degrees C and pH 9.0, and did not require mono- or divalent cations for its activity.
Amino Acid Sequence↗
Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation.
The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.
Adenosine Triphosphate↗
Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli.
In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k(cat) value and an increment in the K(m) values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.
Allosteric Regulation↗
Glucokinase regulatory protein is associated with mitochondria in hepatocytes.
The association of glucokinase with liver mitochondria has been reported [Danial et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952-956]. We confirmed association of glucokinase immunoreactivity with rat liver mitochondria using Percoll gradient centrifugation and demonstrated its association with the 68 kDa regulatory protein (GKRP) but not with the binding protein phosphofructokinase-2/fructose bisphosphatase-2. Substrates and glucagon induced adaptive changes in the mitochondrial glucokinase/GKRP ratio suggesting a regulatory role for GKRP. Combined with previous observations that GKRP overexpression partially inhibits glycolysis [de la Iglesia et al. (2000) The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10597-10603] these findings suggest that there may be distinct glycolytic pools of glucokinase.
Animals↗
Regulatory mechanisms of chitin biosynthesis and roles of chitin in peritrophic matrix formation in the midgut of adult Aedes aegypti.
In mosquitoes, the peritrophic matrix is formed in response to blood feeding and can be a physical barrier when pathogens ingested with blood meal attempt to reach and transverse the midgut epithelium. The main components of the peritrophic matrix are chitin-biding-domain containing proteins, glycosylated proteins, and chitin fibrils. Chitin is synthesized from fructose-6-phosphate by a series of five enzymatic reactions. We previously found that blood feeding induces transcriptional up-regulation of glutamine: fructose-6-phosphate amidotransferase-1 (AeGfat-1) and chitin synthase (AeCs), the first and last enzymes of the biosynthetic pathway, respectively, in the midgut of Aedes aegypti. In this study, we demonstrated that formation of the peritrophic matrix is disrupted when the transcript abundance of either gene is knocked-down using RNAi methodologies. We also have shown that enzymatic activity of recombinant AeGFAT-1 is sensitive to feedback inhibition by UDP-N-acetylglucosamine, a substrate of chitin synthase. These findings demonstrate that in the midgut of adult Ae. aegypti, (1) chitin is synthesized de novo in response to blood feeding and is an essential component of the peritrophic matrix, and (2) chitin biosynthesis is negatively regulated, in part, by inhibitory sensitivity of AeGFAT-1 to UDP-N-acetylglucosamine.
Aedes↗
Effects of brazilin on the production of fructose-2,6-bisphosphate in rat hepatocytes.
Increased hepatic glucose output is one of the major mechanisms of hyperglycemia in diabetic patients. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. Brazilin, an active component of sappan wood (Caesalpinia sappan), decreases blood glucose in diabetic animals. In this study, the effect of brazilin on gluconeogenic intermediate production and enzyme activity were examined to investigate the hypoglycemic mechanism of brazilin. Brazilin increased the production of F-2,6-BP in hepatocytes by elevating intracellular levels of fructose-6-phosphate (F-6-P) and hexose-6-phosphate (H-6-P). Brazilin was also found to significantly increase the activity of 6-phosphofructo-2-kinase (PFK-2) and pyruvate kinase in glucagon-treated hepatocytes. However, glucose-6-phosphatase activity was not affected by brazilin. This data suggests that brazilin inhibits hepatic gluconeogenesis by elevating the F-2,6-BP level in hepatocytes, possibly by elevating cellular F-6-P/H-6-P levels and PFK-2 activity. Increased pyruvate kinase activity may also play a role in the anti-gluconeogenic action of brazilin.
Animals↗
The crystal structure of mouse phosphoglucose isomerase at 1.6A resolution and its complex with glucose 6-phosphate reveals the catalytic mechanism of sugar ring opening.
Phosphoglucose isomerase (PGI) is an enzyme of glycolysis that interconverts glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) but, outside the cell, is a multifunctional cytokine. High-resolution crystal structures of the enzyme from mouse have been determined in native form and in complex with the inhibitor erythrose 4-phosphate, and with the substrate glucose 6-phosphate. In the substrate-bound structure, the glucose sugar is observed in both straight-chain and ring forms. This structure supports a specific role for Lys518 in enzyme-catalyzed ring opening and we present a "push-pull" mechanism in which His388 breaks the O5-C1 bond by donating a proton to the ring oxygen atom and, simultaneously, Lys518 abstracts a proton from the C1 hydroxyl group. The reverse occurs in ring closure. The transition from ring form to straight-chain substrate is achieved through rotation of the C3-C4 bond, which brings the C1-C2 region into close proximity to Glu357, the base catalyst for the isomerization step. The structure with G6P also explains the specificity of PGI for glucose 6-phosphate over mannose 6-isomerase (M6P). To isomerize M6P to F6P requires a rotation of its C2-C3 bond but in PGI this is sterically blocked by Gln511.
Animals↗
The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.
Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea. Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate. To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined. Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues. These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs. These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate.
Archaeal Proteins↗
Computational study of human phosphomannose isomerase: Insights from homology modeling and molecular dynamics simulation of enzyme bound substrate.
Phosphomannose isomerase is a zinc metalloenzyme that catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate, and the three-dimensional (3D) structure of human phosphomannose isomerase has not been reported. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of mannose-6-phosphate isomerase from (PDB code 1PMI). The model structure is further refined by energy minimization and molecular dynamics methods. The mannose-6-phosphate-enzyme complex is developed by molecular docking and the key residues involved in the ligand binding are determined, which will facilitate the understanding of the action mode of the ligands and guide further genetic studies. Our results suggest a hydride transfer mechanism of alpha-hydrogen between the C1 and C2 positions but do not support the cis-enediol mechanism. The detailed mechanism involves, on one side, Zn2+ mediating the movement of a proton between O1 and O2, and, on the other side, the hydrophobic environment formed in part by Tyr278 promoting transfer of a hydride ion.
Amino Acid Sequence↗