scholarly journals The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G

1987 ◽  
Vol 246 (1) ◽  
pp. 83-88 ◽  
Author(s):  
C R Goward ◽  
M D Scawen ◽  
T Atkinson
Keyword(s):  
Enzyme Activity ◽  
Binding Site ◽  
Kinetic Data ◽  
Bacillus Stearothermophilus ◽  
Reactive Dye ◽  
Affinity Probe ◽  
High Concentrations ◽  
Triazine Dye

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.

scholarly journals Kinetics of concomitant transfer and hydrolysis reactions catalysed by the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61

1973 ◽  
Vol 135 (3) ◽  
pp. 483-492 ◽  
Author(s):  
J.-M. Frère ◽  
J.-M. Ghuysen ◽  
H. R. Perkins ◽  
M. Nieto
Keyword(s):  
Enzyme Activity ◽  
Binding Site ◽  
Water Content ◽  
Diaminopimelic Acid ◽  
Acceptor Molecule ◽  
Competitive Inhibitors ◽  
Hydrolysis Pathway ◽  
High Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of

When Ac2-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac2-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac2-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac2-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

scholarly journals Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

1999 ◽  
Vol 46 (2) ◽  
pp. 419-429 ◽  
Author(s):  
M Danieluk ◽  
R Buś ◽  
S Pikuła ◽  
J Bandorowicz-Pikuła
Keyword(s):  
Binding Site ◽  
Binding Proteins ◽  
Direct Sequencing ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Dependent Manner ◽  
Cibacron Blue ◽  
Annexin Vi ◽  
Cibacron Blue 3Ga ◽  
Triazine Dye

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.

scholarly journals Amino acids important in enzyme activity and dimer stability for Drosophila alcohol dehydrogenase

1995 ◽  
Vol 308 (2) ◽  
pp. 419-423 ◽  
Author(s):  
S W Chenevert ◽  
N G Fossett ◽  
S H Chang ◽  
I Tsigelny ◽  
M E Baker ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Alcohol Dehydrogenase ◽  
Binding Site ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Side Chain ◽  
Wild Type ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Substrate Binding Site

We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

scholarly journals Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 25 ◽  
Author(s):  
Xue Yang ◽  
Jinchi Wei ◽  
Zhihai Wu ◽  
Jie Gao
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Abiotic Stress ◽  
Binding Site ◽  
Stress Responses ◽  
Stress Resistance ◽  
Substrate Binding ◽  
Biochemical Properties ◽  
Biotic And Abiotic Stress ◽  
Substrate Binding Site

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

Inhibitory Action of Glufosinate on Photosynthesis

1993 ◽  
Vol 48 (3-4) ◽  
pp. 369-373 ◽  
Author(s):  
Aloysius Wild ◽  
Christine Wendler
Keyword(s):  
Enzyme Activity ◽  
Calvin Cycle ◽  
Atmospheric Conditions ◽  
Direct Inhibition ◽  
Carboxylase Activity ◽  
Bisphosphate Carboxylase ◽  
Transamination Reaction ◽  
Ribulosebisphosphate Carboxylase ◽  
High Concentrations ◽  
Respiratory Conditions

Glufosinate (phosphinothricin) irreversibly blocks the glutamine synthetase which subsequently gives rise to an accumulation of ammonium and to a strong decrease in some amino acids, especially glutamine and glutamate.Under atmospheric conditions (400 ppm CO2, 21% O2) glufosinate causes a rapid inhibition of photosynthesis, too. H ow ever, under non-photo respiratory conditions (1000 ppm CO2, 2% O2) only a slight inhibition of photosynthesis occurs with glufosinate. Since under both conditions an accumulation of ammonium occurs, it is concluded that inhibition of photosynthesis is not induced by the higher concentrations of ammonium. The results rather suggest that the absence of amino donors in the glycolate pathway leads to a break-down of the transamination reaction of glyoxylate to glycine. This causes an inhibition of photorespiration and as a further consequence the inhibition of photosynthesis. There are two hypotheses for explaining this phenomenon. One of them supposes that the blockade in the glycolate pathway produces a lack of Calvin cycle intermediates which subsequently is the cause of the inhibition of photo synthesis. The other one suggests a direct inhibition of the ribulose-1,5-bisphosphate carboxylase by the accumulation of glyoxylate and P-glycolate.After treatment with different intermediates of the Calvin cycle and photorespiration to gether with glufosinate no decrease in the inhibition of photosynthesis could be measured. This suggests that the inhibition of photosynthesis is not induced by a depletion of intermediates of the Calvin cycle.Tests on the effect of glyoxylate and P-glycolate on ribulosebisphosphate carboxylase activity showed that in crude leaves extracts the enzyme activity can only be inhibited by high concentrations of these substances. However, in intact spinach chloroplasts the enzyme activity can be blocked by using much lower concentrations of glyoxylate. This may indicate that the ribulosebisphosphate carboxylase activase is affected by this metabolite and that this may be the reason for an inhibition of photosynthesis after treatment with glufosinate.

Direct Measurement of Iodine Production by Sonic Extracts of Polymorphonuclear Leukocytes

1971 ◽  
Vol 17 (5) ◽  
pp. 392-396 ◽  
Author(s):  
Lawrence R DeChatelet ◽  
Charles E McCall ◽  
M Robert Cooper
Keyword(s):  
Absorption Spectrum ◽  
Kinetic Data ◽  
Polymorphonuclear Leukocytes ◽  
Enzymatic Reaction ◽  
Human Neutrophils ◽  
Enzyme Kinetic ◽  
Ph Optimum ◽  
Beef Liver ◽  
Free Iodine ◽  
High Concentrations

Abstract We describe an enzymatic reaction between iodide ion, H2O2, and neutrophil sonicates, in which free iodine is formed. Some characteristics of the reaction are: (a) it is catalyzed by sonic extracts of human neutrophils, by purified horseradish peroxidase, or purified human myeloperoxidase, but not by sonic extracts of rabbit alveolar macrophages or beef liver catalase; (b) iodine is the product, as shown by its absorption spectrum and the absorption spectrum of the starch adduct; (c) the reaction is proportional to the amount of neutrophil sonicate added, and has a pH optimum near 4.0. Reaction is not linear with respect to time, owing to denaturation of the enzyme. Kinetic data indicate that the enzyme may be allosteric with respect to iodide and is inhibited by high concentrations of H2O2. These represent possible sources of control of the reaction.

scholarly journals High pressures increase α-chymotrypsin enzyme activity under perchlorate stress

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Stewart Gault ◽  
Michel W. Jaworek ◽  
Roland Winter ◽  
Charles S. Cockell
Keyword(s):  
High Pressure ◽  
Enzyme Activity ◽  
Phase Space ◽  
High Pressures ◽  
Extreme Environments ◽  
Combined Effects ◽  
Deep Subsurface ◽  
Subsurface Environments ◽  
Dissolved Ions ◽  
High Concentrations

Abstract Deep subsurface environments can harbour high concentrations of dissolved ions, yet we know little about how this shapes the conditions for life. We know even less about how the combined effects of high pressure influence the way in which ions constrain the possibilities for life. One such ion is perchlorate, which is found in extreme environments on Earth and pervasively on Mars. We investigated the interactions of high pressure and high perchlorate concentrations on enzymatic activity. We demonstrate that high pressures increase α-chymotrypsin enzyme activity even in the presence of high perchlorate concentrations. Perchlorate salts were shown to shift the folded α-chymotrypsin phase space to lower temperatures and pressures. The results presented here may suggest that high pressures increase the habitability of environments under perchlorate stress. Therefore, deep subsurface environments that combine these stressors, potentially including the subsurface of Mars, may be more habitable than previously thought.

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