The nature and reactivity of the ‘essential’ thiol in rabbit muscle creatine kinase III (EC 2.7.3.2)

Rabbit muscle creatine kinase III (EC 2.7.3.2) can be reacted with 2-chloromercuri-4-nitrophenol and this results in the incorporation of two moles of mercurial per mole of enzyme subunit in a biphasic reaction. The second-order rate constant for the slow reaction is 475 ± 42 M^?1 s^?1. S-Carboxamidomethyl-creatine kinase reacts with a single mole of mercurial per mole of subunit. The rate constant, 466 ± 57 M^?1 s^?1, is almost identical to that for the slow reaction of the native enzyme. The reaction between 3-carboxy-4-nitrophenylthio-creatine kinase and 2-chloromercuri-4-nitrophenol has a second-order rate constant of 449 ± 56 M^?1 s^?1. The results may be explained if the mercurial reacts very rapidly with that cysteine residue which reacts independently with iodoacetamide or 5,5′-dithiobis(2-nitrobenzoic acid). However, 2-chloromercuri-4-nitrophenol also reacts more slowly with a second cysteine residue. Definition of the essentiality of thiol groups in enzymes by reaction with labile ligands, here represented by organomercurials, clearly must be approached with caution.     

On the Reaction of Superoxide With DMPO/*OOH

A kinetic model has been used to estimate the rate constant for the reaction of superoxide (O₂/OOH) with the superoxide spin adduct of 5.5-dimethylpyrroline-N-oxide. DMPO/OOH. This rate constant is estimated to be 4.9 (± 2.2) × 10⁶ M^?1 s^?1, pH 7.4 and 25°C.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

1,3‐Propanediol adsorption on a cation exchange resin: Adsorption isotherm,thermodynamics, and mechanistic studies

1,3‐Propanediol (1,3‐PD) is widely used in cosmetics, foods, antifreezes, and polyester. A low‐cost cation exchange resin, 001×7 H‐form resin, was selected for 1,3‐PD adsorption obtained from microbial fermentation of crude glycerol. The thermodynamics and kinetics of adsorption were studied. To identify the characteristics of the adsorption process, the influence of 1,3‐PD concentration, temperature, and resin particle diameters was studied. The temperature dependence of the adsorption equilibrium in the range of 288 to 318 K was observed to satisfy the Langmuir isotherm well. The thermodynamic parameters, adsorption enthalpy, entropy, and Gibbs free energy, were determined as 36.2 kJ·moL^?1, 160 mol^?1·K^?1, and ?11.2 kJ·moL^?1, respectively, which indicated that the adsorption was spontaneous and endothermic. The adsorption kinetics was accurately represented by the shell progressive model and indicated that the particle diffusion was the rate‐limiting step. Based on the kinetic simulation, the rate constant of exchange (k₀), order reaction (α), and the apparent activation energy reaction (E_a) were obtained as 3.11×10^?3, 0.644, and 11.5 kJ·moL^?1, respectively. This kinetic and thermodynamic analysis of 1,3‐PD recovery presented in this article is also applicable for the separation of other polyols by resin adsorption, which will promote value‐added utilization of glycerol.     

Thermodynamics and kinetics of co-operative protein-nucleic acid binding: II. Studies on the binding between protamine and calf thymus DNA

Unspecific binding of a protamine, namely fluorescein-labelled clupeine Z, to double-stranded calf thymus DNA was studied using fluorescence titration methods and chemical relaxation techniques. Both equilibrium and kinetic data have been analysed using general theoretical approaches discussed in the accompanying paper. The results agree well with the predictions made on the basis of a standard co-operative binding model.Basic parameters evaluated are the co-operative binding constant (K), the coefficient measuring co-operative interaction between nearest neighbours (q), the number of nucleotides occupied by one protamine molecule (n) and the rate constant of dissociation at the ends of bound ligand sequences (K_D). Values obtained at 20 °C, pH 7.5 and 0.4 m-NaCl were K = 5.8 × 10⁷m^?1, q = 1700, n = 20 and K_D = 0.29 s^?1. They have been found to be sensitive to the concentration of added salt (NaCl). This effect apparently reflects the essentially electrostatic nature of the binding process. The results can be satisfactorily described in terms of competitive binding of sodium ions.     

The inhibition of activated bovine coagulation factors X and VII by antithrombin III

The inhibition of activated bovine Factors VII and X by antithrombin III has been studied by kinetic methods. The reaction between Factor X_a and antithrombin III is characterized by second-order kinetics, with a rate constant of 3.9 × 10³m^?1s^?1 at pH 7.5 at 37 °C. Inhibition in the presence of excess antithrombin III does not proceed to completion: The decay of Factor X_a deviates from pseudo-first-order kinetics and a final equilibrium is reached, suggesting reversibility. The apparent association constant, at pH 7.5, 37 °C, is 2.3 × 10⁹m^?1. The interaction of three forms of bovine Factor VII with antithrombin III has been studied by the same methods. Factor VII and the two-chain activated form, α-Factor VII_a, and the tissue factor-Factor VII_a complex are not significantly inhibited by plasma levels of antithrombin III, in the either the presence or absence of heparin.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Abstract

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6?±?1 × 10⁷ M^?1 s^?1 at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7?±?3 × 10⁷ M^?1 s^?1 at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK_a of phenolic hydroxyl dissociation in tyrosine is ～ 10.3, this infers a much lower rate constant, about 3 × 10⁵ M^?1 s^?1, for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

The Gibbs Free Energy Threshold for the Invasion of a Microbial Population Under Kinetic Constraints

Possible chemotrophic metabolism at a site of interest is controlled not only by the catabolic energy expressed as the Gibbs energy of reaction (Δ_rG) but also by the kinetic constraints due to the availability of electron acceptors and donors. We introduced graphical and stochastic approaches for determining the Δ_rG threshold required to support a microbial population with a specific catabolic strategy under kinetic constraints. Invasibility as an indicator of the present reproductive ability of a microbial population was evaluated by simultaneously calculating Δ_rG for the catabolic reaction and the microbial catalytic rate. For example, the neutrophilic iron-oxidizing bacteria's invasibility was calculated by randomly choosing the Fe²⁺ and O₂ concentrations between 10^?8 and 10^?2 mol L^?1, and pH between 4 and 8, to determine the Δ_rG threshold for invasion. Parameters were estimated from batch experiments of neutrophilic iron-oxidizing bacteria reported in previous studies. Under the given conditions, the stochastic approach predicted that the neutrophilic iron-oxidizing bacteria can always invade a system in which the Δ_rG for Fe oxidation is below ?90 kJ mol Fe^?1, can occasionally invade if Δ_rG is between ?45 and ?90 kJ mol Fe^?1, and can never invade if Δ_rG is above ?45 kJ mol Fe^?1. The Δ_rG threshold for invasion is sifted by the growth yield coefficient, the loss rate of cells, the maximum cell-specific Fe oxidation rate constant, and the temperature. The Δ_rG threshold for invasion may be unable to rigorously predict the stable dominance of microbial metabolism, but can provide a rough indication for the possible microbial metabolism under current conditions.     

Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin.

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state.Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10⁷m^?1s^?1 and 60 s^?1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0.Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s^?1) and a combination constant which is of the same order of magnitude as reported for undissociated protein (k_on = 4.6 × 10⁷m^?1s${}^{\mathrm{?1}}$). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state.The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.     

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

The Reaction of no With Superoxide

The rate constant for the reaction of NO with ·O₂^? was determined to be (6.7 ± 0.9) × 10⁹ 1 mol^?1 s^?1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O₂^? and the formation of the product ^?OONO. The decay rate of ^?OONO, in the presence of 0.1 moll^?1 formate, ranges from 1.2s^?1 at pH 5 to about 0.2s^?1 in strong base, the latter value probably reflecting catalysis by formate.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Suicide-Peroxide inactivation of microperoxidase-11: A kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H₂O₂. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H₂O₂]>>[AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, α_i, and the apparent inactivation rate constant, k_i, were obtained as 0.282 ± 0.006 min^{? 1} and 0.497 ± 0.013 min^{? 1} at [H₂O₂] = 1.0 mM, 27°C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Catalyses by polymer complexes. VII. Enhanced esterolytic reactivity of polymer-coenzyme A,glutathione complexes

The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′_a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, K_CoASH = 1110–2270 M^?1, K_GSH = 170–503M^?1, k′_a,bound at pH 8.65 = 142–341M^?1 sec^?1. k′_a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′_a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant.     

Superoxide dismutase: a comparison of rate constants

O₂^?was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂^? was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂^? by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂^? with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m^?1 sec^?1 at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂^? in the 10^?8m → 10^?13m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂^? concentrations in the 10^?5m range. The second order rate constant for the enzymatic dismutation of O₂^? is thus independent of the concentration of O₂^? in the range 10^?5 → 10^?13m.Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k′₁[AE] + k″₁[AE][OH^?]^0.5, where k′₁ = 0.020 ± 0.006 s^?1 and k″₁ = 2.1 ± 0.8 (L/mol)^?0.5 s^?1. Irreversible decomposition of the pseudobase is described by rate = k′₂[AE][OH^?], where k′₂ = 20.1 ± 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.