Steady-State Kinetics of Enzyme Reactions in the Presence of Added Nucleophiles

1972 ◽  
Vol 50 (12) ◽  
pp. 1334-1359 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Specific Binding ◽  
Specific Binding Sites ◽  
Steady State Kinetics ◽  
Special Cases ◽  
Kinetics Of Formation ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Concentration Graphs ◽  
Kinetics Of ◽  
Hydrolysis Of

Many enzyme-catalyzed reactions, such as hydrolyses, give rise to two products P1 and P2 which are formed in different reaction steps. The second product P2 is frequently formed by hydrolysis of an intermediate such as an acyl-enzyme or a phosphoryl-enzyme. An alternative nucleophile N introduced into the system forms an additional product P3. The present paper is concerned with the kinetics of formation of P1, P2, and P3 in the presence of added nucleophiles. A number of alternative mechanisms are considered, and equations are derived for the rates of formation of the three products, and the Michaelis constant, as functions of nucleophile concentration. Graphs are presented showing the variations of these parameters with the concentration of N, for a variety of special cases. Special attention is given to the possibility of specific binding sites for the water and the nucleophile molecules.The data for a number of enzyme systems are discussed with reference to the treatment. For reactions catalyzed by alkaline phosphatase it is concluded that only one mechanism (mechanism VI) is consistent with the results.

Cyclization and acid-catalyzed hydrolysis of O-benzoylbenzamidoximes

1986 ◽  
Vol 51 (12) ◽  
pp. 2786-2797
Author(s):  
František Grambal ◽  
Jan Lasovský
Keyword(s):  
Reaction Rate ◽  
Kinetic Isotope ◽  
Acid Base Equilibrium ◽  
Mineral Acids ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Ph Scale ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Derivatives Of

Kinetics of formation of 1,2,4-oxadiazoles from 24 substitution derivatives of O-benzoylbenzamidoxime have been studied in sulphuric acid and aqueous ethanol media. It has been found that this medium requires introduction of the Hammett H0 function instead of the pH scale beginning as low as from 0.1% solutions of mineral acids. Effects of the acid concentration, ionic strength, and temperature on the reaction rate and on the kinetic isotope effect have been followed. From these dependences and from polar effects of substituents it was concluded that along with the cyclization to 1,2,4-oxadiazoles there proceeds hydrolysis to benzamidoxime and benzoic acid. The reaction is thermodynamically controlled by the acid-base equilibrium of the O-benzylated benzamidoximes.

Studies of the uptake of macromolecules by cells. I. Uptake of proteins by cells of the Ehrlich–Lettré ascites carcinoma

1968 ◽  
Vol 46 (12) ◽  
pp. 1443-1450 ◽  
Author(s):  
Y. C. Choi ◽  
E. R. M. Kay
Keyword(s):  
Nucleic Acid ◽  
Cell Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Protein Uptake ◽  
Specific Binding Sites ◽  
Model Protein ◽  
Uptake Process ◽  
Kinetics Of ◽  
Protein Treatment

The uptake of protein by cells of the Ehrlich–Lettré ascites carcinoma was characterized kinetically by using hemoglobin as a model protein. An attempt was made to show that the process is not an artefact due to nonspecific adsorption of protein to the cell membrane. The kinetics of the uptake process suggested that an interaction exists between the exogenous protein and specific binding sites on the membrane. Acetylation of hemoglobin enhanced the rate of uptake of this protein. Treatment of cells with neuraminidase, phospholipase A, and Pronase resulted in an inhibition of protein uptake. The experimental evidence for the uptake of hemoglobin was supported by evidence that L-serine-U-14C-labelled hemoglobin is transported into the cytoplasm and utilized subsequently, resulting in labelling of the nucleic acid nucleotides.

Steady-state and pre-steady-state kinetics of the mitochondrial F(1)F(o) ATPase: is ATP synthase a reversible molecular machine?

2000 ◽  
Vol 203 (1) ◽  
pp. 41-49 ◽  
Author(s):  
A.D. Vinogradov
Keyword(s):  
Steady State ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Gamma Subunit ◽  
Change Mechanism ◽  
Steady State Kinetics ◽  
Energy Dependent ◽  
Variable Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of

H(+)-ATP synthase (F(1)F(o) ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F(1), which is ideally suited for the alternating binding change mechanism, with a rotating gamma-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (P(i)) and Mg(2+) participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F(1) and F(1)F(o) demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, P(i), Mg(2+) and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F(1)F(o) ATPase is proposed.

Enzymic colorimetric determination of phosphatidylglycerol in amniotic fluid.

1984 ◽  
Vol 30 (4) ◽  
pp. 534-537 ◽  
Author(s):  
J D Artiss ◽  
M W McGowan ◽  
D R Strandbergh ◽  
E Epstein ◽  
B Zak
Keyword(s):  
Amniotic Fluid ◽  
Fetal Lung ◽  
Aqueous Sample ◽  
Colorimetric Determination ◽  
Glycerol Phosphate ◽  
Ionic Detergent ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Hydrolysis Of

Abstract We describe a procedure for the enzymic, colorimetric determination of phosphatidylglycerol in amniotic fluid. After extraction into chloroform:methanol (2:1 by vol) and evaporation, the phospholipid-containing residue is redissolved in a non-ionic detergent, which thus provides an aqueous sample. The subsequent enzymic reaction sequence involves phospholipase-catalyzed hydrolysis of glycerol from its phospholipid. Subsequent enzyme-catalyzed reactions phosphorylate this glycerol and oxidize the resulting glycerol phosphate to produce hydrogen peroxide, which is reacted to produce an intense red chromogen in the peroxidase-catalyzed coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate. When used in conjunction with previously reported enzymic techniques for determination of lecithin and sphingomyelin, this procedure may provide an accurate and precise "lung profile" for assessment of fetal lung maturity.

scholarly journals Thermodynamics and Kinetics of Glycolytic Reactions. Part I: Kinetic Modeling Based on Irreversible Thermodynamics and Validation by Calorimetry

2020 ◽  
Vol 21 (21) ◽  
pp. 8341
Author(s):  
Kristina Vogel ◽  
Thorsten Greinert ◽  
Monique Reichard ◽  
Christoph Held ◽  
Hauke Harms ◽  
...  
Keyword(s):  
Equilibrium State ◽  
Kinetic Models ◽  
Metabolic Pathways ◽  
Irreversible Thermodynamics ◽  
Titration Calorimetry ◽  
Metabolic Network Analysis ◽  
Arrhenius Relation ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Kinetics Of

In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride

1977 ◽  
Vol 55 (1) ◽  
pp. 19-26 ◽  
Author(s):  
R. James Maguire
Keyword(s):  
Steady State ◽  
Trichoderma Viride ◽  
Solution Temperature ◽  
Temperature Range ◽  
A Value ◽  
Steady State Kinetics ◽  
Decreasing Ph ◽  
Ph Curve ◽  
Kinetics Of ◽  
Hydrolysis Of

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol–buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 × 10−3 M to 9 × 10−2 M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate – pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol−1 over the temperature range 5–56 °C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol.The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-β-D-glucoside was examined by pre-steady-state methods in which [Formula: see text], and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 s−1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism [Formula: see text]) (ii) a log rate–pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5–6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol−1 at pH 5.7 over the temperature range 10–60 °C.

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