Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

1990 ◽  
Vol 68 (6) ◽  
pp. 965-972 ◽  
Author(s):  
Yuchiong Hsuanyu ◽  
H. Brian Dunford
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Second Order ◽  
Prostaglandin H Synthase ◽  
Compound I ◽  
Stabilizing Agents ◽  
Order Rate ◽  
Ability To Act ◽  
Prostaglandin H ◽  
Compound Ii

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 °C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant K2,app, was found to fall within the range of 5.8 × 106 ± 0.4 × 106 M−1∙s−1 < K2,app < 1.8 × 107 ± 0.1 × 107 M−1∙s−1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme–substrate complex formation, with kcat = 22 ± 3 s−1, Km = 67 ± 10 μM, and the second-order rate constant k3,app = 2.0 × 105 ± 0.2 × 105 M−1∙s−1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 ± 0.5 s−1, Km = 49 ± 7 μM, and k3,app = 1.03 × 105 ± 0.07 × 105 M−1∙s−1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 ± 5 and 0.52 ± 0.05 s−1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 ± 5 and 0.33 ± 0.02 s−1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate. It is suggested that kinetic studies on this enzyme can be performed in the presence of diethyldithiocarbamate, if one is cognizant of the rate of reaction of the stabilizing agent with compounds I and II and corrects for it if necessary.Key words: prostaglandin H synthase, diethyldithiocarbamate, cyclooxygenase, peroxidase, stabilizing agents.

Studies on Horseradish Peroxidase. XI. On the Nature of Compounds I and II as Determined from the Kinetics of the Oxidation of Ferrocyanide

1973 ◽  
Vol 51 (4) ◽  
pp. 582-587 ◽  
Author(s):  
M. L. Cotton ◽  
H. B. Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Rate Constant ◽  
Active Sites ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Compound I ◽  
Order Rate ◽  
Compound Ii ◽  
Kinetics Of

In order to investigate the nature of compounds I and II of horseradish peroxidase, the kinetics were studied of ferrocyanide oxidation catalyzed by these compounds which were prepared from three different oxidizing agents. The pH dependence of the apparent second-order rate constant for ferrocyanide oxidation by compound I, prepared from ethyl hydroperoxide and m-chloroperbenzoic acid, was interpreted in terms of an ionization on the enzyme with a pKa = 5.3, identical to that reported previously for hydrogen peroxide. The second-order rate constant for the compound II-ferrocyanide reaction also showed the same pH dependence for the three oxidizing substrates. However, with more accurate results, the compound II-ferrocyanide reaction was reinterpreted in terms of a single ionization with pKa = 8.5. The same dependence of ferrocyanide oxidation on pH suggests structurally identical active sites for compounds I and II prepared from the three different oxidizing substrates.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Interactions of Heparin and a Covalently Linked Antithrombin Heparin Complex with the Components of the Fibrinolytic Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2211-2211
Author(s):  
Ankush Chander ◽  
Helen M Atkinson ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Serine Proteases ◽  
Conflicts Of Interest ◽  
Order Rate Constant ◽  
Second Order ◽  
Coagulation Cascade ◽  
Simultaneous Addition ◽  
Order Rate ◽  
Significant Difference

Abstract Abstract 2211 Introduction: Unfractionated heparin (UFH) is used for the prophylaxis and treatment of thromboembolic diseases. UFH catalyzes inhibition by antithrombin (AT) of the serine proteases in the coagulation cascade. Additionally, UFH has been shown to interact with components of the fibrinolytic pathway in vitro. However UFH has several limitations which impact its utility as a therapeutic agent. Our lab has developed a novel covalent antithrombin-heparin complex (ATH) which inhibits most serine proteases of the coagulation pathway significantly faster when compared to non covalent mixtures of AT and UFH. However, the interactions of ATH with the components of the fibrinolytic pathway have not been studied before. Thus, the present study investigates possible serpin-heparin interactions of AT + UFH vs ATH within the fibrinolytic pathway. Methods: Discontinuous second order rate constant assays under pseudo-first order conditions were carried out to obtain second order rate constant (k2) values for the inhibition of plasmin by AT+UFH versus ATH. Briefly, at specific time intervals 20 nM plasmin was inhibited by 200 nM AT + 0–5000 nM UFH or by 200 nM ATH in the presence of 2.5 mM Ca2+. Reactions were neutralized by the simultaneous addition of a solution containing polybrene and plasmin substrate S-2366™ in buffer. Residual plasmin activity was measured and the final k2 values calculated. For experiments involving tPA, wells containing 40nM tPA and increasing concentrations of AT, UFH or ATH, at mole ratios ranging from 0 to 20:1, were incubated for 15 min. Reactions with tPA were neutralized by simultaneous addition of a solution containing either polybrene and tPA substrate, S-2288™ in buffer, (ATH and UFH) or only the substrate S-2288™ in buffer (AT). Enzyme activity was then determined by measuring rate of substrate cleavage (Vmax). Results: When plasmin was inhibited by AT in the absence of UFH, k2 values of 2.82×105 +/− 4.46×104 M−1 min−1 were observed. The k2 values increased with addition of successively higher concentrations of UFH up to a plateau with maximal k2 of 5.74×106 +/− 2.78×105 M−1 min−1 at a UFH concentration of 3000nM. For inhibition of plasmin by ATH, k2 values of 6.39 × 106 +/− 5.88 × 105 M−1 min−1 were observed. Inhibition of plasmin by ATH was not significantly different when compared to the highest k2 values obtained with UFH. (p=0.36) No statistically significant difference in tPA enzyme activity was observed when Vmax values for tPA alone were compared with those in the presence of AT, UFH or ATH. (p=0.932, p=0.085, p=0.31 respectively) Significance: The characteristic shape of the curve obtained from the k2vs. UFH plot suggests that the mechanism responsible for inhibition of plasmin by AT+UFH involves conformational activation of the serpin. The k2 values in this study for inhibition of plasmin by both AT+UFH and ATH were three orders of magnitude lower than k2 values for inhibition of thrombin or factor Xa. Furthermore these results suggest that tPA is not inhibited by AT + UFH or ATH, and is not influenced by the presence of UFH alone. Cumulatively, this indicates that the fibrinolytic pathway is minimally impacted by AT + UFH or ATH, allowing maximal antithrombotic potential to be achieved during anticoagulation. Overall, the favourable anticoagulant properties of ATH combined with the findings of this study strengthens the utility of the covalent conjugate over conventional UFH for the treatment of thromboembolic disorders. Disclosures: No relevant conflicts of interest to declare.

Ambident nucleophiles. II. Nitrogen-bonded and carbon-bonded σ-complex formation in the reaction of pyrrolide anions with 1,3,5-trinitrobenzene

1982 ◽  
Vol 60 (15) ◽  
pp. 1988-1995 ◽  
Author(s):  
J. C. Halle ◽  
M. J. Pouet ◽  
M. P. Simonnin ◽  
F. Debleds ◽  
F. Terrier
Keyword(s):  
Aqueous Solution ◽  
Complex Formation ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Reactive Species ◽  
Second Order ◽  
Potassium Salts ◽  
Strong Base ◽  
Order Rate

Reaction of 1,3,5-trinitrobenzene (TNB) with pyrrole, 2,5-dimethyl pyrrole, and 2,4-dimethyl-3-ethyl pyrrole in the presence of a strong base (CH3O−) yields nitrogen- and/or carbon-bonded 1:1 σ-complexes in dimethylsulphoxide (DMSO). Depending on the stoichiometry of the reagents, 1:2 and 2:1 pyrrole–TNB diadducts are also formed. Identification of all complexes was effected by nmr. The reactive species are shown to be the pyrrolide ions and the results emphasize the ambident character of these anions towards an aromatic electrophile. Some of the complexes have been isolated as crystalline potassium salts when experiments are performed in acetonitrile. Among the isolable complexes, the kineticallybutnotthermodynamicallyfavored nitrogen adduct of pyrrole (5a) is remarkably unreactive. The second-order rate constant kH+ for is H+-catalyzed decomposition in aqueous solution is only 1 L mol−1 s−1 (t = 25 °C).

scholarly journals Kinetic studies on the nitrite reductase of Wolinella succinogenes

1986 ◽  
Vol 233 (2) ◽  
pp. 553-557 ◽  
Author(s):  
R Blackmore ◽  
T Brittain
Keyword(s):  
Rate Constant ◽  
Nitrite Reductase ◽  
Flash Photolysis ◽  
Spectral Characteristics ◽  
Order Rate Constant ◽  
Second Order ◽  
Stopped Flow ◽  
Dependent Manner ◽  
Wolinella Succinogenes ◽  
Order Rate

The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 × 106 M-1 × S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.

Formation and spectra of flavin radicals in the presence of β-mercaptoethanol at pH 9 and 10

1984 ◽  
Vol 62 (3) ◽  
pp. 580-585 ◽  
Author(s):  
Parminder S. Surdhar ◽  
Rizwan Ahmad ◽  
David A. Armstrong
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Order Rate Constant ◽  
Second Order ◽  
Radical Species ◽  
Spectral Changes ◽  
Order Rate ◽  
Visible Spectra ◽  
Uv Visible ◽  
Presence And Absence

Spectral changes and rates of reaction of flavins and several radical species have been investigated at pH 7, 9, and 10 in the presence and absence of β-mercaptoethanol. The radicals •CO2−, eaq−, and [Formula: see text] reacted with FAD at pH 10 to give a spectrum of FAD •Fl− with rate constants of 7 ± 1 × 108 and 4 ± 1 × 108 M−1 s−1 for •CO2− and [Formula: see text] respectively. At pH 7 only •FlH was observed and at pH 9 a mixture of •FIH and •Fl−.Interactions between flavin radicals and sulphydryl at 10−4 M concentration did not cause perturbations in the uv–visible spectra until either the radical and/or the sulphydryl were ionized. With FAD at pH 9 or 10 and LFl at pH 10 the 370 nm peak of •Fl− was enhanced by about 15% and a second larger growth occurred near 450 nm in the presence of 10−4 to 10−2 M sulphydryl. We attribute this to the formation of labile intermediate RSHFl•−, which must also be involved in the reduction of Fl by [Formula: see text] at pH 9 or 10.The second order rate constant k13 for reaction of [Formula: see text] with FAD at pH 9 and 10 was found to be 4.2 ± 0.5 × 108 M−1 s−1 and 2.0 ± 0.4 × 108 M−1 s−1 respectively. The rate constant for the reaction between [Formula: see text] and LFl at pH 10 was slightly faster, 7 ± 1 × 108 M−1 s−1, probably reflecting the fact that LFl lacks the bulky negatively charged adenine dinucleotide group of FAD.

Studies on Sulphamic Acid and Related Compounds. II. Kinetics and Mechanism of Hydrolysis of Trimethylamine- and of Triethylamine Sulphur Trioxide Addition Compounds

1962 ◽  
Vol 15 (2) ◽  
pp. 251 ◽  
Author(s):  
BE Fleischfresser ◽  
I Lauder
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Aqueous Acetone ◽  
Second Order ◽  
Fluoride Ions ◽  
Order Rate ◽  
Mechanism Of Hydrolysis ◽  
Reaction With Water ◽  
Sulphamic Acid ◽  
Hydrolysis Of

The kinetics of hydrolysis of trimethylamine- and of triethylaminesulphur trioxide addition compounds have been studied in water and in aqueous acetone. Reaction occurs according to the equation,������������� f - + R,N.SO,+H,O-tR,XH+HSO~.The solvolysis reactions are first-order and are not catalyzed by acids. The halide ions, Cl', Br', and 1', show only a normal salt effect on the rate of hydrolysis of + - (CH,),N.SO, but in the presence of fluoride ions, the rate constant for the production + - of acid from (C,H,),N.SO, in water at 95 OC is about one-seventh of that in the absence of fluoride under the same conditions. It is suggested that the fluorosulphonate ion is formed rapidly, and that this ion then undergoes slow hydrolysis :�In the presence of alkali, using water as the solvent, second-order kinetics are observed, the equation for the reaction being,�������������� + - R,N.SO,+~OH-+R,X+SO~= +H,O. Assuming the reaction with water is bimolecular, the ratio of the (bimolecular) rate constants at 35 OC, ko~-/k~,o is approximately lo8 for each complex. In aqueous acetone, at low water concentrations, the hydrolysis reactions of the trialkylaminesulphur trioxide complexes show second-order kinetics. At 35 OC for the hydrolysis of + - (CH,),N.SO, the ratio of the second-order rate constant in aqueous acetone to the + - (calculated) second-order rate constant in water is approximately 550 ; for (C,H,),N.SO, the same ratio is 6900. It is considered that hydrolysis occurs in water and in aqueous acetone via a bi- molecular attack at sulphur.

On the Interaction Between Human Plasma Kallikrein and C1-Esterase Inhibitor

1983 ◽  
Vol 49 (03) ◽  
pp. 193-195 ◽  
Author(s):  
Torbjörn Nilsson
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Rate Constant ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Second Order ◽  
Plasma Kallikrein ◽  
First Order ◽  
Order Rate ◽  
Esterase Inhibitor

SummaryThe kinetics of the reaction between human plasma kallikrein and CĪ-esterase inhibitor was studied in a purified system. By monitoring the inhibition reaction for extended periods of time, it was found to proceed in two consecutive steps, a fast reversible second-order binding step followed by a slower, irreversible first-order transition. The rate constants in this reaction model were determined, as well as the dissociation constant of the initial, reversible enzyme-inhibitor complex. Thus, at 37° C the second-order rate constant was found to be 5 · 104 M -1 · s-1, the first order rate constant was 5 · 10-4 s-1 and the dissociation constant K was 1.5 · 10-8 M. Heparin (28 U/ml) and 6-aminohexanoic acid (10 mM) had no effect on the k1 of the of the reaction.

Sign in / Sign up

Export Citation Format

Share Document