scholarly journals Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis

1997 ◽  
Vol 328 (1) ◽  
pp. 83-91 ◽  
Author(s):  
A. Eduard SERGIENKO ◽  
D. K. SRIVASTAVA
Keyword(s):  
Experimental Data ◽  
Glycogen Phosphorylase ◽  
Equilibrium Constants ◽  
Glycogen Synthesis ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Minimal Models ◽  
Dimeric Unit ◽  
Catalysed Reaction

We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 μM) and glucose 1- phosphate (G1P; 4 μM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.

scholarly journals Identification of the kinetic mechanism of succinyl-CoA synthetase

2013 ◽  
Vol 33 (1) ◽  
Author(s):  
Xin Li ◽  
Fan Wu ◽  
Daniel A. Beard
Keyword(s):  
Experimental Data ◽  
Catalytic Mechanism ◽  
Model Simulation ◽  
Tricarboxylic Acid Cycle ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Catalysed Reaction ◽  
Dead End ◽  
Haem Biosynthesis ◽  
Parameter Values

The kinetic mechanism of SCS [succinyl-CoA (coenzyme A) synthetase], which participates in the TCA (tricarboxylic acid) cycle, ketone body metabolism and haem biosynthesis, has not been fully characterized. Namely, a representative catalytic mechanism and associated kinetic parameters that can explain data on the enzyme-catalysed reaction kinetics have not been established. To determine an accurate model, a set of putative mechanisms of SCS, proposed by previous researchers, were tested against experimental data (from previous publication) on SCS derived from porcine myocardium. Based on comparisons between model simulation and the experimental data, an ordered ter–ter mechanism with dead-end product inhibition of succinate against succinyl-CoA is determined to be the best candidate mechanism. A thermodynamically constrained set of parameter values is identified for this candidate mechanism.

Combustion Simulation of Propane/Oxygen (With Nitrogen/Argon) Mixtures Using Rate-Controlled Constrained-Equilibrium

2018 ◽  
Vol 141 (2) ◽  
Author(s):  
Guangying Yu ◽  
Hameed Metghalchi ◽  
Omid Askari ◽  
Ziyu Wang
Keyword(s):  
Experimental Data ◽  
Energy System ◽  
Kinetic Mechanism ◽  
Chemical Kinetic ◽  
Oxygen Mixture ◽  
Free Valence ◽  
Wide Range ◽  
Constrained Equilibrium ◽  
Rate Controlled ◽  
Good Agreement

The rate-controlled constrained-equilibrium (RCCE), a model order reduction method, has been further developed to simulate the combustion of propane/oxygen mixture diluted with nitrogen or argon. The RCCE method assumes that the nonequilibrium states of a system can be described by a sequence of constrained-equilibrium states subject to a small number of constraints. The developed new RCCE approach is applied to the oxidation of propane in a constant volume, constant internal energy system over a wide range of initial temperatures and pressures. The USC-Mech II (109 species and 781 reactions, without nitrogen chemistry) is chosen as chemical kinetic mechanism for propane oxidation for both detailed kinetic model (DKM) and RCCE method. The derivation for constraints of propane/oxygen mixture starts from the eight universal constraints for carbon-fuel oxidation. The universal constraints are the elements (C, H, O), number of moles, free valence, free oxygen, fuel, and fuel radicals. The full set of constraints contains eight universal constraints and seven additional constraints. The results of RCCE method are compared with the results of DKM to verify the effectiveness of constraints and the efficiency of RCCE. The RCCE results show good agreement with DKM results under different initial temperature and pressures, and RCCE also reduces at least 60% CPU time. Further validation is made by comparing the experimental data; RCCE shows good agreement with shock tube experimental data.

scholarly journals Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

1988 ◽  
Vol 250 (1) ◽  
pp. 25-31 ◽  
Author(s):  
C MacKintosh ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Intact Cells ◽  
Competing Anions ◽  
Effects Of Ph ◽  
Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

scholarly journals Isomerization of an enzyme-coenzyme complex in yeast alcohol dehydrogenase-catalysed reactions

2003 ◽  
Vol 68 (2) ◽  
pp. 77-84 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Kinetic Mechanism ◽  
Yeast Alcohol Dehydrogenase ◽  
Catalyzed Reactions ◽  
The Individual ◽  
Individual Rate ◽  
Catalyzed Oxidation

In this work, all the rate constants in the kinetic mechanism of the yeast alcohol dehydrogenase-catalyzed oxidation of ethanol by NAD+, at pH 7.0, 25 ?C, have been estimated. The determination of the individual rate constants was achieved by fitting the reaction progress curves to the experimental data, using the procedures of the FITSIM and KINSIM software package of Carl Frieden. This work is the first report in the literature showing the internal equilibrium constants for the isomerization of the enzyme-NAD+ complex in yeast alcohol dehydrogenase-catalyzed reactions.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

scholarly journals Equilibrium Modeling of Astaxanthin Extraction from Haematococcus pluvialis

2021 ◽  
Vol 21 (3) ◽  
pp. 554
Author(s):  
Putri Restu Dewati ◽  
Rochmadi Rochmadi ◽  
Abdul Rohman ◽  
Avido Yuliestyan ◽  
Arief Budiman
Keyword(s):  
Experimental Data ◽  
High Performance ◽  
Haematococcus Pluvialis ◽  
Equilibrium Constants ◽  
Extraction Methods ◽  
Single Step ◽  
Equilibrium Models ◽  
Extraction Equilibrium ◽  
Assisted Extraction ◽  
Solid Liquid

Astaxanthin is a natural antioxidant, and the highest content of this compound is found in Haematococcus pluvialis microalgae. Microwave-assisted extraction (MAE) is one of the environmentally friendly extraction methods and has many advantages. This study aims to investigate the extraction of astaxanthin through the MAE method using various solvents. Several equilibrium models were proposed to describe this solid-liquid equilibrium. The solid-liquid extraction equilibrium parameters were determined by minimizing the sum of squares of errors (SSE), in which equilibrium constants were needed for scaling up purposes. Previously, the microalgae were pretreated with HCl to soften their cell walls in order to improve the extraction recovery. In this study, dichloromethane, acetone, methanol, and ethanol were used as the solvents for extraction. The astaxanthin concentration was determined by high-performance liquid chromatography (HPLC) and spectrophotometry. Astaxanthin was found to attain equilibrium at 57.42% recovery in a single-step extraction. Thus, several steps were required in sequence to obtain an optimum recovery. The experimental data were fitted to three equilibrium models, namely, Henry, Freundlich, and Langmuir models. The experimental data were well fitted to all the models for the extraction in dichloromethane, methanol, ethanol and acetone, as evident from the almost same SSE value for each model.

Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands

1990 ◽  
Vol 259 (5) ◽  
pp. F758-F767
Author(s):  
G. A. Ahearn ◽  
P. Franco
Keyword(s):  
Driving Forces ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Affinity Constant ◽  
Electrical Potential Difference ◽  
Static Head ◽  
Transmembrane Electrical Potential

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.

scholarly journals N-Acetyl-β-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1

1997 ◽  
Vol 328 (2) ◽  
pp. 695-700 ◽  
Author(s):  
Mary BOARD
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Protein Phosphatase 1 ◽  
P Concentration ◽  
Glucose Analogue ◽  
Stimulation Of

Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-β-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 μM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 μM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 μM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented.

scholarly journals Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups

1990 ◽  
Vol 272 (2) ◽  
pp. 359-367 ◽  
Author(s):  
J Rúa ◽  
D de Arriaga ◽  
F Busto ◽  
J Soler
Keyword(s):  
Enzyme Inhibitor ◽  
Isocitrate Lyase ◽  
Equilibrium Constants ◽  
Spectrophotometric Titration ◽  
Kinetic Mechanism ◽  
Thiol Groups ◽  
Phycomyces Blakesleeanus ◽  
Denatured State ◽  
Nitrobenzoic Acid ◽  
Dead End

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5′-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.

Fuels Characterization for Use in Internal Combustion Engines

2001 ◽  
Author(s):  
R. Lanzafame ◽  
M. Messina
Keyword(s):  
Experimental Data ◽  
Wide Temperature Range ◽  
Internal Combustion Engines ◽  
Equilibrium Constants ◽  
Combustion Process ◽  
Internal Combustion ◽  
Least Square ◽  
Mathematical Functions ◽  
Temperature Range ◽  
Ic Engines

Abstract It is important provide mathematical functions able to fit with great precision experimental data on gases properties, in order to obtain reliable results when computerized models on IC engines are used. On the basis of experimental data on equilibrium constants (for dissociation phenomena occurring during combustion process in IC engines) new mathematical functions have been determined to fit experimental data. In comparison to traditional fitting polynomials, these new mathematical functions present a great accuracy in matching experimental data. These new mathematical functions have the functional forms of a V order Logarithmic Polynomial, and their coefficients have been evaluated on the basis of the least square method. The new V order Logarithmic Polynomials have been determined for several dissociation reactions according to internal combustion processes applications. V order Logarithmic Polynomials have been implemented also to describe the trend of specific heat at constant pressure Vs temperature and enthalpy Vs temperature. These new Logarithmic Polynomials have been calculated for several gases and fuels for IC engines applications. The new Logarithmic Polynomials pointed out a better precision in comparison to the others polynomial functions used in literature, and the possibility to utilize a single Logarithmic Polynomial for a wide temperature range, according to a good accuracy with experimental data. Another advantage of the Logarithmic Polynomials is the possibility to extrapolate experimental data on a wide temperature range (25% of experimental T range) in order to supply to the experimental data shortage.

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