scholarly journals The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site

1980 ◽  
Vol 189 (3) ◽  
pp. 561-567 ◽  
Author(s):  
D Roberts ◽  
G L Kellett
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Amp ◽  
Rate Constant ◽  
Catalytic Site ◽  
Stopped Flow ◽  
Diffusion Controlled ◽  
Inhibitory Site ◽  
Kinetics Of ◽  
Step Mechanism ◽  
Effector Binding

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.

scholarly journals Kinetic evidence for a two-step mechanism for the binding of chymotrysin to α1-proteinase inhibitor

1989 ◽  
Vol 259 (3) ◽  
pp. 929-930 ◽  
Author(s):  
M Bruch ◽  
J G Bieth
Keyword(s):  
Reaction Mechanism ◽  
Rate Constant ◽  
Proteinase Inhibitor ◽  
Stopped Flow ◽  
Inhibitor Concentration ◽  
Displacement Method ◽  
First Order ◽  
Flow Apparatus ◽  
Pseudo First Order ◽  
Step Mechanism

We have used the proflavin displacement method and a stopped-flow apparatus to measure the rate constant for the binding of 2 microM-chymotrypsin to 20-125 microM-alpha 1-proteinase inhibitor. The observed pseudo-first-order constant showed a hyperbolic dependence on alpha 1-proteinase inhibitor concentration, suggesting a reaction mechanism in which a fast pre-equilibrium (K = 0.19 mM) is followed by a first-order formation of the final complex (k = 252 s-1).

Kinetics of the Anation Reaction of Nickel(II) and 1,10-Phenanthroline in Ethanol

1972 ◽  
Vol 50 (23) ◽  
pp. 3861-3865 ◽  
Author(s):  
M. L. Sanduja ◽  
W. MacF. Smith
Keyword(s):  
Rate Constant ◽  
Sodium Perchlorate ◽  
Order Rate Constant ◽  
Stopped Flow ◽  
Tetrabutyl Ammonium ◽  
Kinetics Of Formation ◽  
Kinetics Of ◽  
Nickel Perchlorate ◽  
Anation Reaction ◽  
Made In

The kinetics of formation of the monophenanthroline complex of nickel(II) in ethanol has been studied using stopped-flow methods over the temperature range 7 to 35 °C. Tetrabutyl ammonium perchlorale in concentration 0.044 M does not affect the rate appreciably, sodium perchlorate at the same concentration depresses the rate significantly. Most measurements were made in the absence of electrolytes other than nickel perchlorate and a trace of perchloric acid. The second order rate constant is not significantly dependent on the nickel(II) concentration over a four-fold change in value indicating that the concentration of encounter pairs is small relative to the concentration of the free reactants. The rate constant at 25 °C (31 × 103 M−1 s−1)is consistent with a dissociative interchange mechanism and the rate constant for ethanol exchange on nickel. However, the value of ΔH≠ for the overall reaction (15.9 ± 1.0 kcal mol−1) is about 5 kcal mol−1 higher than that reported for ethanol exchange.

Etude du système acridine orange – poly(C) et de son interaction avec les complexes du palladium(II)

1984 ◽  
Vol 62 (9) ◽  
pp. 1681-1686 ◽  
Author(s):  
Robert Ménard ◽  
Miklos Zador
Keyword(s):  
Acridine Orange ◽  
Thermodynamic Parameters ◽  
Rate Constant ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Flow Method ◽  
Polycytidylic Acid ◽  
Low Concentrations ◽  
C Complex ◽  
Kinetics Of

The complex formed between acridine orange (AO) and polycytidylic acid (poly(C)) was studied by spectrophotometry and spectrofluorometry. The complex was characterized by its stoichiometry, structure, and the thermodynamic parameters of its formation. The results are in agreement with an external aggregation of the protonated dye along the negatively charged poly(C) chain and indicate that approximately two AO molecules are bound per nucleotide unit of poly(C). The kinetics of the reaction between this complex and a Pd(II) complex was studied by the stopped-flow method. The addition of (dien)Pd(II) to the AO–poly(C) complex leads to the dissociation of the latter, due to fixation of the Pd(II) complex to the N3 site of the cytosine base of poly(C). The rate constant for the AO liberation, extrapolated at zero AO concentration, corresponds to the rate constant of Pd(II) fixation on poly(C). This indicates that AO can be used as an indicator for this reaction and allows kinetic studies at very low concentrations (≤ 5 × 10−6 M).

The formation and decay of the oxyferrous complex of beef adrenocortical cytochrome P-450scc. Rapid-scan and stopped-flow studies

1987 ◽  
Vol 65 (5) ◽  
pp. 486-492 ◽  
Author(s):  
Mohammed A. Kashem ◽  
H. Brian Dunford
Keyword(s):  
Rate Constant ◽  
Acid Group ◽  
Spontaneous Decay ◽  
Stopped Flow ◽  
Oxygen Binding ◽  
First Order Kinetics ◽  
Rapid Scan ◽  
Ph Range ◽  
Kinetics Of ◽  
Cytochrome P 450

The formation and spontaneous decay of the oxyferrous complex of purified beef adrenocortical cholesterol-bound (high spin) cytochrome P-450scc have been studied by means of rapid-scan spectrometry in the Soret region at 4 °C. The oxyferrous complex, the formation of which occurs within 40 ms with a Soret absorption peak at 422 nm, is unstable and decays spontaneously to the ferric cholesterol-bound cytochrome P-450scc. The rapid-scan spectra for both processes were recorded. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous complex at 418 nm, between oxyferrous complex and ferric cytochrome P-450scc at 411 nm. The kinetics of oxygen binding and spontaneous decay of the oxyferrous complex have also been studied at 4 °C by means of stopped-flow experiments in the pH range 5.1–8.8. The rate constant for oxygen binding is constant at 5.8 × 105 ± 0.8 × 105 M−1∙s−1 over the pH range of the study. On the other hand, the decay process exhibited pH-dependent monophasic first-order kinetics. The rate constant for the decay appears to be influenced by an acid group with a pKa of 7.1 on the oxyferrous complex of cholesterol-bound cytochrome P-450scc.

Acid dependence and ion pairing in the anation reactions yielding iron(III) monosulfate

1970 ◽  
Vol 48 (19) ◽  
pp. 3100-3103 ◽  
Author(s):  
F. L. Baker ◽  
W. MacF. Smith
Keyword(s):  
Rate Constant ◽  
Ion Pairing ◽  
Ion Pair ◽  
Pair Formation ◽  
Association Constants ◽  
Stopped Flow ◽  
Ion Pair Formation ◽  
Significant Dependence ◽  
Kinetics Of ◽  
Acid Dependence

Stopped-flow studies of the kinetics of the a nation reaction yielding iron(III) monosulfate indicate a significant dependence of rate constant on acidity and also a dependence on iron concentrations. This is consistent with association constants for outer ion pair formation between the reactants which have about three times the Fuoss values.

scholarly journals Stopped-Flow Spectrophotometric Studies of the Kinetics of Interaction of Dihydroxyfumaric Acid with the DPPH Free Radical

2010 ◽  
Vol 5 (2) ◽  
pp. 83-87
Author(s):  
Natalia Secara
Keyword(s):  
Free Radical ◽  
Reaction Kinetics ◽  
Rate Constant ◽  
Stopped Flow ◽  
Flow Method ◽  
Reaction Proceeds ◽  
Reaction Orders ◽  
Stoichiometric Reaction ◽  
Two Stages ◽  
Kinetics Of

The reaction of dihydroxyfumaric acid with the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied using the stopped-flow method, in order to describe the reaction kinetics. Dihydroxyfumaric acid reacts very rapidly with DPPH, the reaction being completed in several minutes. This 2-stoichiometric reaction proceeds in two stages, with reaction orders of 1 and 0.76 with respect to DPPH, and 0.5 and 0.3 with respect to DHF, respectively. The rate constant of the two stages of the reaction were found to be 39.1 (L/mol•s) and 0.0012 (s-1) at 20º C and pH 4.0.

scholarly journals Temperature-dependences of the kinetics of reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features

2006 ◽  
Vol 396 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Sheraz Gul ◽  
Geoffrey W. Mellor ◽  
Emrys W. Thomas ◽  
Keith Brocklehurst
Keyword(s):  
Conformational Change ◽  
Rate Constants ◽  
Catalytic Site ◽  
Stopped Flow ◽  
Thiol Groups ◽  
Cysteine Peptidases ◽  
Temperature Dependences ◽  
Significant Conformational Change ◽  
Kinetics Of ◽  
Molecular Recognition Features

The temperature-dependences of the second-order rate constants (k) of the reactions of the catalytic site thiol groups of two cysteine peptidases papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) with a series of seven 2-pyridyl disulphide reactivity probes (R-S-S-2-Py, in which R provides variation in recognition features) were determined at pH 6.7 at temperatures in the range 4–30 °C by stopped-flow methodology and were used to calculate values of ΔS‡, ΔH‡ and ΔG‡. The marked changes in ΔS‡ from negative to positive in the papain reactions consequent on provision of increase in the opportunities for key non-covalent recognition interactions may implicate microsite desolvation in binding site–catalytic site signalling to provide a catalytically relevant transition state. The substantially different behaviour of actinidin including apparent masking of changes in ΔH‡ by an endothermic conformational change suggests a difference in mechanism involving kinetically significant conformational change.

scholarly journals The mechanism of addition of pyridoxal 5′-phosphate to Escherichia coli apo-serine hydroxymethyltransferase

2007 ◽  
Vol 404 (3) ◽  
pp. 477-485 ◽  
Author(s):  
Francesca Malerba ◽  
Andrea Bellelli ◽  
Alessandra Giorgi ◽  
Francesco Bossa ◽  
Roberto Contestabile
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Spectral Properties ◽  
Direct Evidence ◽  
Serine Hydroxymethyltransferase ◽  
Stopped Flow ◽  
Cofactor Binding ◽  
Kinetics Of ◽  
Flow Experiments ◽  
Step Mechanism

Previous studies suggest that the addition of pyridoxal 5′-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5′-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.

Kinetics of the Reaction of Nickel(II) and 2,2′-Bipyridine in Ethanol

1973 ◽  
Vol 51 (23) ◽  
pp. 3975-3977 ◽  
Author(s):  
M. L. Sanduja ◽  
W. MacF. Smith
Keyword(s):  
Kinetic Parameters ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Second Order ◽  
Stopped Flow ◽  
Ligand Specificity ◽  
Temperature Range ◽  
Kinetics Of Formation ◽  
The Difference ◽  
Kinetics Of

The kinetics of formation of the monobipyridine complex of nickel(II) in ethanol has been studied with stopped-flow methods over the temperature range 7 to 35 °C. The value of the second order rate constant kf at 25 °C of 6.6 × 10−3M−1 s1 and the values of ΔH≠ (10.1 ± 1.0 kcal mol−1) and of ΔS≠ (−7.3 ± 3.4 cal deg−1 mol−1) are close to the corresponding values for ethanol exchange on nickel(II) and suggest that the mechanism is dissociative interchange. However the difference in the values of the kinetic parameters of this reaction and those previously reported for the reactions involving the chemically similar phenanthroline imply a degree of ligand specificity for the reactions in ethanol which is considerably larger than is the case for reactions in water and methanol and that a common Id mechanism with monodentate formation being rate controlling is not applicable to both reactions.

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