scholarly journals Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei

1987 ◽  
Vol 245 (1) ◽  
pp. 13-18 ◽  
Author(s):  
C N Cronin ◽  
K F Tipton
Keyword(s):  
Trypanosoma Brucei ◽  
Initial Rate ◽  
Reaction Pathway ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Enzyme Concentration ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Fructose 1,6 Bisphosphate ◽  
Kinetics Of

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.

scholarly journals The kinetic mechanism of the major form of ox kidney aldehyde reductase with d-glucuronic acid

1982 ◽  
Vol 205 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ann K. Daly ◽  
Timothy J. Mantle
Keyword(s):  
Glucuronic Acid ◽  
Alternative Pathway ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Aldehyde Reductase ◽  
Major Form ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Inhibition Studies ◽  
Kinetics Of

The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

KINETIC STUDIES OF METHYL-BIS-β-CHLOROETHYLAMINE: III. THE KINETICS OF THE DIMERIZATION IN METHANOL

1948 ◽  
Vol 26b (2) ◽  
pp. 175-180 ◽  
Author(s):  
C. A. Winkler ◽  
A. W. Hay ◽  
A. L. Thompson
Keyword(s):  
Rate Constants ◽  
Initial Rate ◽  
Kinetic Studies ◽  
First Order ◽  
Rate Expression ◽  
Principal Reaction ◽  
Kinetics Of ◽  
Rate Controlling Step

The principal reaction of methyl-bis-β-chloroethylamine in methanol is dimerization, which results in one chlorine from each molecule becoming ionic, but this is accompanied by slight alcoholysis. The rate-controlling step is believed to be the first order formation of an ethylenimonium ion which reacts rapidly with one of its kind to form dimer. The rate expression as calculated from initial rate constants is k (initial) = 4.0 × 1013e−19600/RThr.−1.

Succinyl Coenzyme A Synthetase of Escherichia coli: Initial Rate Kinetics of Succinyl-CoA Cleavage and Isotope Exchange Studies

1973 ◽  
Vol 51 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Frank J. Moffet ◽  
W. A. Bridger
Keyword(s):  
Isotope Exchange ◽  
Coenzyme A ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Constant Ratio ◽  
Rate Kinetics ◽  
Kinetics Of ◽  
Succinyl Coenzyme A Synthetase

Initial rate kinetic studies of succinyl coenzyme A synthetase of E. coli in the direction of succinyl-CoA cleavage are consistent with the operation of a partially random sequential kinetic mechanism with initial binding of ADP followed by random association of succinyl-CoA and Pi. The mechanism is analogous to that proposed previously for the succinyl-CoA formation reaction, and thus the kinetic mechanism of the overall reversible succinyl-CoA synthetase reaction appears to be symmetrical.Studies of the kinetics of [Formula: see text] isotope exchange at equilibrium show that this partially random sequential kinetic mechanism is not an exclusive pathway. [Formula: see text] isotope exchange rates did not show complete substrate inhibition when CoA or succinate was varied in constant ratio with Pi. However, when CoA or succinate was varied in constant ratio with succinyl-CoA, nearly complete substrate inhibition was observed. These results can be interpreted in terms of a wide variety of minor pathways of substrate binding and product release available to the enzyme under various conditions.

scholarly journals Kinetic studies of oxidized nicotinamide–adenine dinucleotide-facilitated reactions of d-glyceraldehyde 3-phosphate dehydrogenase

1974 ◽  
Vol 143 (2) ◽  
pp. 353-363 ◽  
Author(s):  
Patricia J. Harrigan ◽  
David R. Trentham
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Temperature Jump ◽  
Reaction Pathway ◽  
Kinetic Studies ◽  
Rate Equations ◽  
First Order ◽  
Diffusion Controlled ◽  
Relaxation Temperature ◽  
Pig Muscle ◽  
Kinetics Of

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD+ has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8°C the rate of acylation of the NAD+-bound (or holo-) enzyme was 3.3×105m−1·s−1 and the rate of phosphorolysis, the reverse reaction, was 7.5×103m−1·s−1. After a temperature-jump perturbation the equilibrium of NAD+ binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate–apoenzyme complex were ≤40m−1·s−1 and ≤12s−1 respectively at pH8.0 and 22°C, which means that both processes are too slow to contribute significantly to the reaction pathway of the reversible NAD+-linked oxidative phosphorylation of d-glyceraldehyde 3-phosphate. Phosphorolysis of both acyl-apoenzyme and acyl-holoenzyme was first-order in Pi up to 100mm-Pi and more. PO43− could be the reactive species of the phosphorolysis of the acyl-holoenzyme, in which case phosphorolysis is a diffusion-controlled reaction, although other kinetically indistinguishable rate equations for the reaction are possible.

THE SILVER CATALYZED OXIDATION OF ETHYLENE: III. KINETICS OF ETHYLENE OXIDE OXIDATION

1954 ◽  
Vol 32 (4) ◽  
pp. 432-442 ◽  
Author(s):  
A. Orzechowski ◽  
K. E. MacCormack
Keyword(s):  
Ethylene Oxide ◽  
Initial Rate ◽  
Oxidation Mechanism ◽  
Kinetic Studies ◽  
Main Step ◽  
Direct Oxidation ◽  
Flow Rates ◽  
Rate Determining Step ◽  
Kinetics Of ◽  
Catalyzed Oxidation

A flow type apparatus was used for kinetic studies of the silver catalyzed oxidation of ethylene oxide (EtO) by oxygen at 274 °C. Using N2 as diluent the concentrations of O2 and ethylene oxide were varied independently from 9.9 to 79% and 2.35 to 9.4% respectively while a total pressure of 1 atmosphere was maintained. Flow rates were varied to give a range of contact times varying from 0.06 to 0.25 sec. It was shown that EtO is oxidized without previous dissociation into C2H4 and O2. The dependence of the initial rate of oxidation of EtO on reactant concentrations excludes isomerization of EtO (to acetalde hyde) as a main step in its oxidation, and a direct oxidation mechanism is suggested. The results of a few experiments to determine the extent of isomerization of EtO to acetaldehyde in the absence of oxygen are presented. No steady state could be achieved but the results may be used semiquantitatively to support the belief that isomerization is not the rate determining step in the oxidation of ethylene oxide.

scholarly journals Kinetics of the monomer-dimer reaction of yeast hexokinase PI

1992 ◽  
Vol 287 (2) ◽  
pp. 567-572 ◽  
Author(s):  
J G Hoggett ◽  
G L Kellett
Keyword(s):  
Glucose Concentration ◽  
Relaxation Times ◽  
Kinetic Studies ◽  
Relaxation Effect ◽  
Enzyme Concentration ◽  
Diffusion Controlled ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Yeast Hexokinase

Kinetic studies of the glucose-dependent monomer-dimer reaction of yeast hexokinase PI at pH 8.0 in the presence of 0.1 M-KCl have been carried out using the fluorescence temperature-jump technique. A slow-relaxation effect was observed which was attributed from its dependence on enzyme concentration to the monomer-dimer reaction; the reciprocal relaxation times tau-1 varied from 3 s-1 at low concentrations of glucose to 42 s-1 at saturating concentrations. Rate constants for association (kass.) and dissociation (kdiss.) were determined as a function of glucose concentration using values of the equilibrium association constant of the monomer-dimer reaction derived from sedimentation ultracentrifugation studies under similar conditions, and also from the dependence of tau-2 on enzyme concentration. kass. was almost independent of glucose concentration and its value (2 x 10(5) M-1.s-1) was close to that expected for a diffusion-controlled process. The influence of glucose on the monomer-dimer reaction is entirely due to effects on kdiss., which increases from 0.21 s-1 in the absence of glucose to 25 s-1 at saturating concentrations. The monomer and dimer forms of hexokinase have different affinities and Km values for glucose, and the results reported here imply that there may be a significant lag in the response of the monomer-dimer reaction to changes in glucose concentrations in vivo with consequent hysteretic effects on the hexokinase activity.

scholarly journals Kinetic studies of the mechanism of pig kidney aldehyde reductase

1981 ◽  
Vol 193 (2) ◽  
pp. 485-492 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Initial Rate ◽  
Kinetic Studies ◽  
Random Order ◽  
Alcohol Oxidation ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Aldehyde Reduction

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

scholarly journals Kinetic studies with the low-Km aldehyde reductase from ox brain

1985 ◽  
Vol 227 (2) ◽  
pp. 621-627 ◽  
Author(s):  
C M Ryle ◽  
K F Tipton
Keyword(s):  
Initial Velocity ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Binary Complex ◽  
Aldehyde Reductase ◽  
Apparent Dissociation Constant ◽  
Displacement Mechanism ◽  
Sequential Mechanism ◽  
Inhibition Studies

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

scholarly journals Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase

1977 ◽  
Vol 167 (3) ◽  
pp. 787-798 ◽  
Author(s):  
Roy D. Waight ◽  
Paul Leff ◽  
William G. Bardsley
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Rate Equation ◽  
Substrate Concentration ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Flip Flop ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Recent Classification

1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis–Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/ν) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis–Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.

Cytoplasmic malate dehydrogenase from Phycomyces blakesleeanus: Kinetics and mechanism

1985 ◽  
Vol 63 (10) ◽  
pp. 1097-1105 ◽  
Author(s):  
Francisco Teixido ◽  
Dolores De Arriaga ◽  
Félix Busto ◽  
Joaquin Soler
Keyword(s):  
Malate Dehydrogenase ◽  
Ternary Complex ◽  
Initial Rate ◽  
Potassium Phosphate ◽  
Product Inhibition ◽  
Potassium Phosphate Buffer ◽  
Phycomyces Blakesleeanus ◽  
Coenzyme Binding ◽  
The Individual ◽  
Inhibition Studies

The kinetics and reaction mechanism of cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) in 0.1 M potassium phosphate buffer (pH 7.5) at 30 °C have been investigated. The initial rate and product inhibition studies were consistent with an ordered bi-bi mechanism that involved more than one kinetically significant ternary complex and also with the coenzyme binding first. The dissociation of the coenzyme from the enzyme–coenzyme complex appeared to be the slowest step in either direction of the reaction. The kinetic and rate constants for the individual steps of the reaction were determined.

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