scholarly journals Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain

1989 ◽  
Vol 257 (2) ◽  
pp. 541-548 ◽  
Author(s):  
P R Young ◽  
A V Briedis
Keyword(s):  
Decanoic Acid ◽  
Kinetic Mechanism ◽  
Order Rate Constant ◽  
Bovine Brain ◽  
Glutathione S Transferase ◽  
Rate Acceleration ◽  
Hydrophobic Binding ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed.

Detailed kinetics of hydrogen abstraction from trans-decalin by OH radicals: the role of hindered internal rotation treatment

2020 ◽  
Vol 22 (44) ◽  
pp. 25740-25746
Author(s):  
Tam V.-T. Mai ◽  
Lam K. Huynh
Keyword(s):  
Master Equation ◽  
Hydrogen Abstraction ◽  
Kinetic Mechanism ◽  
Oh Radicals ◽  
Rate Model ◽  
Wide Range ◽  
Detailed Kinetics ◽  
Kinetics Of ◽  
Detailed Kinetic Mechanism

The detailed kinetic mechanism of the trans-decalin + OH reaction is firstly investigated for a wide range of conditions (T = 200–2000 K & P = 0.76–76000 Torr) using the M06-2X/aug-cc-pVTZ level and stochastic RRKM-based Master equation rate model.

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

1986 ◽  
Vol 64 (7) ◽  
pp. 681-691 ◽  
Author(s):  
Jose E. Gonzalez ◽  
Ronald L. Somerville
Keyword(s):  
Feedback Inhibition ◽  
Kinetic Mechanism ◽  
Ternary Complexes ◽  
Hill Coefficient ◽  
Anthranilate Synthase ◽  
Active Centers ◽  
Catalytic Sites ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Tryptophan Molecule

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme–anthranilate–pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accomodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 μM) and phosphoribosylpyrophosphate (Km = 50 μM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 μM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod–Wyman–Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregrate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.

scholarly journals MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

1974 ◽  
Vol 61 (2) ◽  
pp. 514-536 ◽  
Author(s):  
David L. Brown ◽  
G. Benjamin Bouck
Keyword(s):  
Protein Synthesis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Short Term ◽  
Protein Pool ◽  
Microtubule Protein ◽  
Kinetics Of ◽  
Mitotic Inhibitor ◽  
Kinetics Of Inhibition

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

Kinetic Studies on Oxidation of Veratryl Alcohol by Laccase-Mediator System. Part 1. Effects of Mediator Concentration

Holzforschung ◽  
2000 ◽  
Vol 54 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Mikhail Yu. Balakshin ◽  
Chen-Loung Chen ◽  
Josef S. Gratzl ◽  
Adrianna G. Kirkman ◽  
Harald Jakob
Keyword(s):  
Kinetic Studies ◽  
Veratryl Alcohol ◽  
Alcohol Oxidation ◽  
Order Rate Constant ◽  
Molar Ratio ◽  
Active Centers ◽  
First Order ◽  
System Part ◽  
Kinetics Of

Summary Kinetics of the laccase-catalyzed oxidation of veratryl alcohol with dioxygen in the presence of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt (ABTS), the mediator, were studied to elucidate the possible reaction mechanism and the role of the mediator in this reaction. The reaction follows a pseudo-first order reaction law. The first order rate constant (κ) is dependent on the Mediator/Substrate (M/S) ratio and has a maximum at M/S molar ratio of 0.15. The kinetic studies show that the mechanism of veratryl alcohol oxidation with dioxygen-laccase-ABTS is rather complex and includes different reaction pathways. The mediator is involved in competitive reactions. It has been suggested that at low mediator concentration, the veratryl alcohol is oxidized via the laccase redox cycle. The mediator acts mostly as a laccase activator at a M/S ratio lower than 0.15. With increasing ABTS concentration with respect to the substrate concentration, ABTS acts increasingly as a cosubstrate competing with the original substrate for active centers of the laccase. This results in inhibition of veratryl alcohol oxidation in the enzyme cycle and increases the role of substrate oxidation by an oxidized mediator.

scholarly journals Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa

1989 ◽  
Vol 262 (2) ◽  
pp. 651-658 ◽  
Author(s):  
M F Scully ◽  
V Ellis ◽  
N Shah ◽  
V Kakkar
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Order Rate Constant ◽  
Pseudo First Order Reaction ◽  
High Affinity ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP

2008 ◽  
Vol 417 (1) ◽  
pp. 355-360 ◽  
Author(s):  
Jingsong Yang ◽  
Francesca Zappacosta ◽  
Roland S. Annan ◽  
Kelvin Nurse ◽  
Peter J. Tummino ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Specific Activity ◽  
Kinetic Mechanism ◽  
Aurora B ◽  
Peptide Substrate ◽  
Catalytic Role ◽  
Substrate Phosphorylation ◽  
Kinetics Of ◽  
Loop Residue

Aurora kinases are a family of serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. AurB (Aurora B kinase) has shown a clear link to cancer and is being pursued as an attractive cancer target. Multiple small molecules targeting AurB have entered the clinic for the treatment of cancer. A protein cofactor, INCENP (inner centromere protein), regulates the cellular localization and activation of AurB. In the present study, we examined the effect of INCENP on the activation kinetics of AurB and also elucidated the kinetic mechanism of AurB-catalysed substrate phosphorylation. We have concluded that: (i) substoichoimetric concentrations of INCENP are sufficient for AurB autophosphorylation at the activation loop residue Thr232, and hence INCENP plays a catalytic role in AurB autophosphorylation; (ii) AurB/INCENP-catalysed phosphorylation of a peptide substrate proceeds through a rapid equilibrium random Bi Bi kinetic mechanism; and (iii) INCENP has relatively minor effects on the specific activity of AurB using a peptide substrate when compared with its role in AurB autoactivation. These results indicate that the effects of INCENP, and probably accessory proteins in general, may differ when enzymes are acting on different downstream targets.

ASSOCIATION OF HEPARIN AND FACTOR Xa: INFLUENCE ON THE RATE OF INHIBITION OF FACTOR Xa BY ANTITHROMBIN III-HEPARIN

1987 ◽  
Author(s):  
B A Owen ◽  
W G Owen
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Order Rate Constant ◽  
Buffer System ◽  
Gel Chromatography ◽  
First Order Kinetics ◽  
Internal Volume ◽  
Two Peaks ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.

scholarly journals Ca2+-dependent high-affinity complex formation between calmodulin and melittin

1983 ◽  
Vol 209 (1) ◽  
pp. 269-272 ◽  
Author(s):  
M Comte ◽  
Y Maulet ◽  
J A Cox
Keyword(s):  
Complex Formation ◽  
Gel Filtration ◽  
Peptide Hormones ◽  
Bovine Brain ◽  
Disc Electrophoresis ◽  
Hydrophobic Drugs ◽  
Basic Peptide ◽  
High Affinity ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.

Effects of the Nucleophile Structure on the Mechanisms of Reaction of 1-Chloro-2,4-dinitrobenzene with Aromatic Amines in Aprotic Solvents

1999 ◽  
Vol 64 (10) ◽  
pp. 1583-1593 ◽  
Author(s):  
Norma Sbarbati Nudelman ◽  
Cecilia E. Silvana Alvaro ◽  
Monica Savini ◽  
Viviana Nicotra ◽  
Jeannette Yankelevich
Keyword(s):  
Hydrogen Bond ◽  
Tertiary Amine ◽  
Aromatic Amines ◽  
Order Rate Constant ◽  
Aprotic Solvents ◽  
Nucleophilic Substitutions ◽  
Hydrogen Bond Acceptor ◽  
Mixed Aggregates ◽  
Kinetics Of

The kinetics of reactions of 1-chloro-2,4-dinitrobenzene with aniline and several substituted aromatic amines, B, in toluene shows a quadratic dependence of the second-order rate constant, kA, on [B], which is preserved even in the presence of increasing amounts of dimethylaniline, while the reaction with N-methylaniline shows a linear dependence of kA vs [B]. All these results are interpreted by the "dimer nucleophile" mechanism, and confirmed by the effects of a non-nucleophilic hydrogen bond acceptor tertiary amine which show the relevance of the structure of the nucleophile and the role of mixed aggregates in defining the mechanisms of aromatic nucleophilic substitutions with amines in aprotic solvents.

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