bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII
Authors: ROSE Matthias , ALBIG Werner , ENTIAN Karl‐Dieter 
Published: March 2005

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102–246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C‐terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression.To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose‐phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double‐null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose‐phosphorylating activity, which is threefold increased compared to wild‐type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.
Properties of the elongation factor 1α in the thermoacidophilic archaebacterium Sulfolobus solfataricus
Authors: MASULLO Mariorosario , RAIMO Gennaro , PARENTE Augusto , GAMBACORTA Agata , DE ROSA Mario , BOCCHINI Vincenzo 
Published: March 2005

The elongation factor 1α (aEF‐1α) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE‐Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF‐1α is the counterpart of the eubacterial elongation factor Tu (EF‐Tu).Purified aEF‐1α is a monomeric protein with a relative molecular mass of 49 000 as determined by SDS/PAGE and by gel filtration on Sephadex G‐100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF‐Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF‐1α from Artemia salina or of archaebacterial EF‐1α from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen‐bond‐forming or non‐helix‐forming residues suggest that common structural features exist among the factors compared.aEF‐1α shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95°C; it is also quite heat‐resistant, since after a 6‐h exposure at 60°C and 87°C the residual [3H]GDP‐binding ability was still 90% and 54% of the control, respectively.The affinity of aEF‐1α for GDP and GTP was also evaluated. At 80°C Ka′ for GDP was about 30‐fold higher than Ka′ for GTP; at the same temperature Kd′ for GDP was 1.7 μM and Kd′ for GTP was 50 μM; these values were 300‐fold and 100‐fold higher, respectively, than those reported for E. coli EF‐Tu at 30°C; compared to the values at 0°C of EF‐Tu from E. coli and T. thermophilus or EF‐1α from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors. At 80°C k′−1 GDP and k′+1 GDP of aEF‐1α were 7.8 min−1 and 4.6 min−1·μM−1, respectively; comparison with the values attributed to E. coli EF‐Tu at 30°C indicates that the dissociation rate constant increases and the association rate constant decreases, thus indicating that the higher Kd′ for GDP of aEF‐1α is due to a synchronous alteration of the rates of both the dissociation and the association processes between GDP and aEF‐1α.Besides aEF‐1α, the other two elongation factors, aEF‐2 and aEF‐1β were also present in S. solfataricus, as suggested by the occurrence of reactions typical of each of them. This allows the hypothesis that in this extremophilic archaebacterium the protein synthesis process follows the same general pattern found in all other living organisms.
Two‐dimensional 1H‐NMR studies of phospholipase‐A2‐inhibitor complexes bound to a micellar lipid‐water interface
Authors: DEKKER Niek , PETERS Anton R. , SLOTBOOM Arend J. , BOELENS Rolf , KAPTEIN Robert , DIJKMAN Ruud , DE HAAS Gerard 
Published: March 2005

One‐ and two‐dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid‐water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two‐dimensional NOE spectra recorded for the enzyme‐inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase‐A2‐micelle complex: (i) the 38‐kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle‐binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)‐dodecyl‐2‐aminohexanol‐1‐phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd≤ 10 μM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical‐shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (< 0.4 nm) near Phe5, Ile9, Phe22 and Phe106.Comparison of our results on the enzyme‐inhibitor‐micelle ternary complex with the crystal structure of the enzyme‐inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. & Verheij, H. M. (1990) Nature 347, 689–691] shows that the mode of inhibitor binding is similar.
Isoforms of human cytochrome‐c oxidase
Authors: VAN KUILENBURG André B. P. , DEKKER Henk L. , VAN DEN BOGERT Coby , NIEBOER Popko , VAN GELDER Bob F. , MUIJSERS Anton O. 
Published: March 2005

The subunit pattern and the steady‐state kinetics of cytochrome‐c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome‐c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus‐encoded subunits of the various cytochrome‐c oxidase preparations.Tissue homogenates, in which cytochrome‐c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome‐c oxidase steady‐state kinetics. Cytochrome‐c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady‐state reaction between human ferrocytochrome c and the four human cytochrome‐c oxidase preparations revealed large differences for the low‐affinity TNmax (maximal turnover number) value, ranging from 77 s−1 for kidney to 273 s−1 for liver cytochrome‐c oxidase at pH 7.4, I= 18 mM. It is proposed that the low‐affinity kinetic phase reflects an internal electron‐transfer step. For the steady‐state reaction of human heart cytochrome‐c oxidase with human cytochrome c, Km and TNmax values of 9 μM and 114 s−1 were found, respectively, at high ionic strength (I= 200 mM, pH 7.4). Only minor differences were observed in the steady‐state activity of the various human cytochrome‐c oxidases.The interaction between human cytochrome‐c oxidase and human cytochrome‐c proved to be highly specific. At high ionic strength, a large decrease in steady‐state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady‐state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome‐c oxidase deficiency.
Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor‐resistant mutants
Authors: TRON Thierry , CRIMI Massimo , COLSON Anne‐Marie , DEGLI ESPOSTI Mauro 
Published: March 2005

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non‐polar residues in a highly conserved region of the protein.The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129 → Leu is not greatly affected either in its ubiquinol: cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147 → Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular‐dichroic spectrum of the low‐potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.
Glycosphingolipids in insects
Authors: HELLING Friedhelm , DENNIS Roger D. , WESKE Bianka , NORES Gustavo , PETER‐KATALINIC Jasna , DABROWSKI Ursula , EGGE Heinz , WIEGANDT Herbert 
Published: March 2005

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two‐dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica‐gel column chromatography and characterized by the presence of a N‐acetyl‐glucosamine‐bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast‐atombombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing‐4‐enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as:(PEtn‐6′)GlcNAc(β1–3)Man(β1–4)GlcβCer;GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc (β1–3)Man(β1–4)GlcβCer;Gal(β1–3)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;Gal(β1–3)GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;GlcNAc(β1–3)Gal(β1–3)GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer.
Conformational dynamics and solvent viscosity effects in carboxypeptidase‐A‐catalyzed benzoylglycylphenyllactate hydrolysis
Authors: GOGUADZE Nina G. , HAMMERSTAD‐PEDERSEN Jan M. , KHOSHTARIYA Dimitrij E. , ULSTRUP Jens 
Published: March 2005

We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 2H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular friction‐free nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site.We illustrate the approach by new spectrophotometric and pH‐titration data for carboxypeptidase‐A‐catalyzed benzoylglycyl‐l‐phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 2H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180–240‐nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme‐peptide substrate complex with a ‘tight’ structure protected from the solvent. In comparison, the enzyme‐ester substrate complex is ‘softer’, strongly coupled to the solvent, and the rate‐determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.
Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators
Authors: SCHNELL Norbert , ENTIAN Karl‐Dieter 
Published: March 2005

o‐Phenanthroline (1,10‐phenanthroline) is a chemical known to chelate iron and other transition metal ions. This compound was added to solid yeast media to reduce the concentration of biologically available iron. Other essential divalent cations, like Zn2+ or Cu2+, which could also be bound, were supplemented. Growth of wild‐type yeast strains was totally inhibited at specific concentrations of the chelator. However, several cells containing plasmids of a multicopy vector genomic library of S. cerevisiae could be selected by growth on these media. All of the resistant clones carried a single additional gene, PAR1 on their multicopy plasmids.Plasmid‐directed overexpression of PAR1 increased the resistance of transformants to o‐phenanthroline and additionally conferred resistance to 1‐nitroso‐2‐naphthol, an iron(III)‐binding molecule with different coordinating ligands. By supplementing the o‐phenanthroline‐containing media with several different metal ions, it could be proved that the selection plates really caused a specific iron limitation. These observations clearly demonstrated that the overexpressed PAR1 gene enables the cell to compete with iron‐chelating organic molecules. PAR1 null mutants, constructed by insertion of the LEU2 gene into the open reading frame, showed a remarkable phenotype: they did not grow on slightly alkaline buffered media (pH > 7) and became hypersensitive to oxidative stress by hydrogen peroxide. Of several heavy metal ions, such as Fe3+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+, tested for supplementation of the alkaline growth deficiency, only iron, either added in the ferrous or ferric form, was able to restore cellular growth.It can be concluded from the DNA sequence that PAR1 encodes a highly acidic protein of 650 residues with mostly hydrophilic character. Some interesting repetitive amino acid motifs, such as (Asp‐Asn)4 or Cys‐Ser‐Glu, may act as metal‐binding sites. The possible role of PAR1 is discussed.
Competitive inhibition of liver glucokinase by its regulatory protein
Authors: VANDERCAMMEN Annick , VAN SCHAFTINGEN Emile 
Published: March 2005

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half‐saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl‐CoA were synergistic with that by N‐acetyl‐glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl‐CoA were competitive with each other, indicating that these two inhibitors bind to the same site.The regulatory protein exerted a non‐competitive inhibition with respect to Mg‐ATP at concentrations of this nucleotide <0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6‐phosphate.The following anions inhibited glucokinase non‐competitively with respect to glucose: Pi, sulfate, I−, Br−, NO−3, Cl−, F− and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6‐phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I− >Br− > NO3− > Cl − > F− acetate and their effect was non‐competitive with respect to fructose 6‐phosphate.Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl‐CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low‐Km specific glucokinase from Bacillus stearothermophilus.
Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation
Authors: DETHEUX Michel , VANDERCAMMEN Annick , VAN SCHAFTINGEN Emile 
Published: March 2005

In the absence of fructose 6‐phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15‐fold) lower potency than in the presence of a saturating concentration of fructose 6‐phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6‐phosphate.In the presence of regulatory protein, sorbitol 6‐phosphate, a potent analog of fructose 6‐phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6‐phosphate versus 1/[sorbitol 6‐phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6‐phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6‐phosphate versus 1/[sorbitol 6‐phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1‐phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half‐maximal effect increased linearly with the concentrations of sorbitol 6‐phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase.Sorbitol 6‐phosphate, 2‐deoxysorbitol 6‐phosphate and mannitol 1‐phosphate, all analogs of the open‐chain configuration of fructose 6‐phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6‐phosphate, whereas fixed analogs of the furanose form of fructose 6‐phosphate were inactive or behaved as activators. This indicated that fructose 6‐phosphate in its open‐chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1‐phosphate, psicose 1‐phosphate, ribitol 5‐phosphate, analogs of fructose 1‐phosphate and of ribitol 5‐phosphate and, at much higher concentrations, inorganic phosphate.
A fluorescence spectroscopic study of glutaminyl‐tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism
Authors: BHATTACHARYYA Tista , BHATTACHARYYA Anusree , ROY Siddhartha 
Published: March 2005

Interaction between Escherichia coli glutaminyl‐tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAGln and ATP bind with dissociation constants of 460, 0.22 and 180 μM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme‐bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5′‐(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive‐ligand binding. tRNA also only has a weak effect on phosphodiester binding.Selectively pyrene‐labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNAGln crystal structure indicates that no major change in shape and size occurs upon tRNAGln binding to GlnRS. 5,5′‐Bis(8‐anilino‐1‐naphthalene sulfonate) (bis‐ANS), a non‐covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNAGln binding.We conclude that lack of tRNA‐independent pyrophosphate‐exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.