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1.
Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ? [S]0 and [S]0 ? [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant (k2Ks)max = 4.2 × 107 M?1 s?1. The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10?5 m, 360 ± 15 s?1 and 29.3 ± 0.8 s?1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and Km of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a k?1k2 value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.  相似文献   

2.
The rates of electron exchange between ferricytochrome c (CIII)3 and ferrocytochrome c (CII) were observed as a function of the concentrations of ferrihexacyanide (FeIII) and ferrohexacyanide (FeII) by monitoring the line widths of several proton resonances of the protein. Addition of FeII to CIII homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, CIII·FeII, in the over-all reaction:
CIII+FeII?k?1k1CIII·FeII?k?2k2CII·FeIII?k?3k3CIII+FeII
Values for k1k?1 = 0.4 × 103m?1and k2 = 208 s?1, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of CIII in the presence of FeII and either KCl or FeIII suggest that complexation is principally ionic, that FeIII and FeII compete for a common site. Addition of saturating concentrations of FeIII to CIII produced only minor changes in the nuclear magnetic resonance spectrum of CIII suggesting that complexation occurs on the protein surface.Addition of FeIII to CII in the presence of excess FeII (to retain most of the protein as CII) increased the line width of the methyl protons of ligated methionine 80. A value for k?2 ≈ 2.08 × 104 s?1 was calculated from the dependence of linewidth on the concentration of FeII at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k2 and k?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k2 and k?2, respectively.  相似文献   

3.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m.  相似文献   

4.
The rate of reaction of [Cr(III)Y]aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO3)] at various temperatures. The general rate equation, Rate = k1 + k2K1[H+]?11 + K1[H+]?1 [Cr(III)Y]aq[H2O2] holds over the pH range 5–9. The decomposition reaction of H2O2 is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k1 and k2 were found to be 1.75 × 10?2 M?1 s?1 and 0.174 M?1 s?1 at 303 K respectively, where k2 is the rate constant for the aquo species and k2 is that for the hydroxo complex. The respective activation enthalpies (ΔH*1 = 58.9 and ΔH*2 = 66.5 KJ mol?1) and activation entropies (ΔS*1 = ?85 and ΔS*2 = ?40 J mol?1 deg?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK1, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).  相似文献   

5.
The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation k′2 = kbKa(Ka + [H+]) + ka[OH?]Ka(Ka + [H+]), where Ka is the dissociation constant of TrisH+. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of kb and ka terms to k2. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the kb term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 102 and possibly of the order 106. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.  相似文献   

6.
The kinetic parameters for the hydrolyses of different l-α-amino acid-β-naphthylamides by Bacillus subtilis aminopeptidase have been measured for the native enzyme and for the enzyme activated in 5 mm Co(NO3)2. In most cases Co2+ activation decreased Km(app) values and increased kcat values, in other cases km(app) and kcat values were increased; for the remainder of the substrates tested km(app) values and kcat values were decreased. In all cases tested the ratios of (kcatKm(app))CO2+/(kcatKm(app)nativ) were increased (2- to 108-fold). For the native enzyme the order of specificity toward the l-amino acid-β-naphthylamides was Arg > Met > Trp > Lys > Leu and for the Co2+ activated enzyme the order of specificity was Lys > Arg > Met > Trp > Leu. The native enzyme hydrolyzed Pro-β-naphthylamide, but not α-Glu-β-naphthylamide; Co2+ activation of the enzyme affected an appreciable rate of hydrolysis of the latter substrate.  相似文献   

7.
A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states QA?QB and QAQB?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of α = [QA?QB]([QA?QB] + [QAQB?]) = 0.065 ± 0.005 (T = 21°C, pH 8).  相似文献   

8.
A theoretical model which can account for both the dynamic and steady responses is proposed based on the occupation theory. The reaction scheme used is;
Here, S and A are stimulus chemicals and receptor sites unbound, respectively. The binding of S to A leads to an active complex (SA)active, which is successively transformed into an inactive complex (SA)active. The response is assumed to be proportional to number of (SA)active. When a stimulating solution is applied instantaneously at t = 0, the solution to the set of differential equations based on the above scheme is obtained as follows;
p=α1e1t22t+ Ck?1k1+(1+k2k?2)C
where p and C stand for the fraction of (SA)active to the total number of receptor sites and stimulus concentration, respectively, and αi, and ωi (i = 1, 2) are numerical parameters depending on the rate constants and on C. The steady response is expressed as the third term in the above equation, which indicates that the response accords with the Beidler taste equation. Mathematical analysis of the above scheme shows that the dynamic response appears when k1C > k?2, and the calculated results for the dynamic response agree approximately with the Hill equation. The Hill coefficient lays within 1·00 and 0·79 and reaches unity with increasing k?1k2, which implies that the dynamic response under this condition satisfies the Beidler taste equation. For the case of gradual application of stimuli, i.e. the experimental condition, the time course of p is simulated with use of an analogue computer rather than with a numerical solution to the above equation. The results indicate that the dynamic response diminishes with decreasing the application speed of stimulus solution. The present theory accounts consistently for various experimental data observed in the chemoreceptor systems.  相似文献   

9.
Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH2CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are kcatKm = 5.04 × 104 min?1M?1, decreasing with either acid or base (PKE1 = 5.64, pKE2 = 9.55), and kcat = 1.02 × 102 min?1, decreasing with acid (pKES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl.  相似文献   

10.
An explicit set of general methods for the experimental determination of the rates k1 and k2 of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity kenv = k1k2(k1 + k2) is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?kenvt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of κ = k2k1. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.  相似文献   

11.
A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: (4.5±1.4) · 103s?1 and (6.9±1.2) · 103 s?1, respectively.The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7±0.8) · 103 s?1.The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.  相似文献   

12.
13.
The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log (C?β) = constant + (ΔH12.3RT) + (ΔW1el2.3RT) ? K′ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. ΔH1 and ΔW1el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), Ks is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A1 (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A1 was assumed to be in equilibrium with D at the same time that it is in equilibrium with A2, A3, etc., dimers and trimers, etc., of A1 in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, ΔWel1/T was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C ? β) versus 1/T is linear with a slope of ΔH1/2.3R. The results fit such an equation over nearly a 20 °C-temperature range with a single value of ΔH1 of +32 kcal/mol A1. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of ΔW1el of 1.22 kcal/mol A1 at pH 6.7 and a value of 4.93 for Ks. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A1 protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A1 before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H+ ions must be bound per A1 or 0.5 per protein polypeptide chain. This amounts to 1 H+ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H+ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.  相似文献   

14.
A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, PQAhv P+Q?A and PQAQBhv P+QAQ?B, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and QA and QB are ubiquinones. In reaction centers containing only QA, the enthalpy of P+Q?A is very close to that of the PQA ground state (ΔHr = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P+QAQ?B in reaction centers containing both quinones has a ΔHr of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q?A and QB, ΔHr for forming P+Q?AQB is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔHr values given above were measured under conditions that minimized this influence. Although the reductions of QA and QB involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P+ and Q?A or Q?B.  相似文献   

15.
16.
N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O2 dependent and shows general catalysis by H2PO4? (k1 = 2.3 M?2 sec?1) and PO4?3 (k2 = 2.3 × 105M?2 sec?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.  相似文献   

17.
The dephosphorylation of ADP and ATP was characterized as the first-order rate constant in dependence on pH in the absence and presence of Cu2+, and together with Cu2+ and a second ligand. The reaction is strongly accelerated by Cu2+ and passes through pH optima at about 6.2 and 6.5 for the Cu2+ ?ADP and ?ATP systems, respectively (I = 0.1, NaClO4; 50°C). In the presence of 2,2′-bipyridyl (Bipy), ternary complexes are formed with the nucleotides ADP or ATP (NP), Cu(Bipy)(NP), which are very stable towards dephosphorylation over a large pH range. Similar stabilizing effects were observed in ternary complexes formed with imidazole or OH?. These results can easily be rationalized by taking into account that in the binary Cu2+ complexes macrochelates are formed by the interaction between the adenine moiety and the metal ion. This interaction is crucial for obtaining the labile species and hence, in the mixed-ligand complexes, where the macrophelate can not be formed, the phosphates are protected toward hydrolysis. In agreement with these results is the dephosphorylation behavior of Cu(CDP)? and Cu(CTP)2?; they are rather stable. This is in accord with the small coordination tendency of the cytosine moiety.By computing the pH dependence of the distribution of the several species, it is shown that the active species are Cu(ATP)2? and Cu(ADP)? and not the hydroxy complexes, [Cu(ATP)(OH)]26? and [Cu(ADP)(OH)24? as were suggested earlier. With the aid of the initial rate, ν0 = d[PO43?]dt, the rate laws of the ascending side of the pH optima were determined: ν0 = k[Cu(NP)][H+]. The descending side of the pH optima is attributed to the formation of Cu(NP)(OH), where the metal ion interaction with N-7 of the adenine moiety is inhibited.  相似文献   

18.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation kn = ko (1 ? n + nπa1)4 with πa1 ~ 0.74, where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and πa1 is the isotopic fractionation factor.  相似文献   

19.
The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O2? and CO2? was determined in H2O and in 2H2O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with kH2Ok2H2O = 1.25 to 1.85, depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.  相似文献   

20.
A group of n susceptible individuals exposed to a contagious disease isconsidered. It is assumed that at each point in time one or more susceptible individuals can contract the disease. The progress of this simple batch epidemic is modeled by a stochastic process Xn(t), t∈[0, ∞), representing the number of infectiveindividuals at time t. In this paper our analysis is restricted to simple batch epidemics with transition rates given by 2Xn(t){n ?Xn(t) +Xn(0)}]12, t∈[0, ∞), α∈(0, ∞). This class of simple batch epidemics generalizes a model used and motivated by McNeil (1972) to describe simple epidemic situations. It is shown for this class of simple batch epidemics, that Xn(t), with suitable standardization, converges in distribution as n→∞ to a normal random variable for all t∈(0, t0), and t0 is evaluated.  相似文献   

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