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1.
Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst. 相似文献
2.
Ter-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation has been measured by oxygen consumption and malonaldehyde (MDA) formation. It has been found that the singlet oxygen (1O2) trap 2,5 diphenylfuran depressed both oxygen consumption and MDA formation. In contrast, histidine, another 1O2 trap does not effect neither oxygen consumption, nor MDA production. On the other hand, β-carotene, a 1O2 quencher strongly depresses oxygen consumption but slightly affects MDA production. Such results are consistent with the generation of 1O2 as transient by product of peroxidative microsomal lipid decomposition. 相似文献
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Enzymatic reaction intermediates of horseradish peroxidase, compounds I and II, were studied by high-resolution nuclear magnetic resonance spectroscopy at 220 MHz. The heme peripheral proton peaks were successfully obtained in the downfield region of 50 to 80 ppm from 4,4-dimethyl-4-silapentane-5-sulfonate for compound I and of 10 to 20 ppm for compound II at pH 9.2. This indicates that no isoporphyrin appears in the catalytic cycle of the enzyme. Temperature dependences of the spectra also were determined for these compounds between 7 and 32 degrees C. With increasing temperature, all the peaks in the downfield region for compound I shifted upfield, obeying the Curie law. These results suggest that the Fe atoms in compounds I and II are in ferryl high- and low-spin states, respectively. The spectrum was also observed in solutions of horse metmyoglobin to which hydrogen peroxide (H2O2) was added. The electron formulations of the hemes in their spectra. Evidence was found against a pi-cation radical on the heme ring as a source of the oxidizing equivalent in compound I. 相似文献
5.
The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I. 相似文献
6.
In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH. 相似文献
7.
Transient-state reactions of horseradish peroxidase compounds I and II with 1-methoxy-4-(methylthio)benzene (a para-substituted thioanisole) were studied over the pH range from 3.4 to 10.5. The pH-jump technique was applied to the compound II reactions at pH values below 8.6. The reactions of both compound I and compound II with the para-substituted thioanisole consisted predominantly of an initial burst. The burst was followed by a steady-state phase that became more obvious at lower concentrations of the thioanisoles. The burst phase for both compounds I and II can be explained in terms of two independent transient-state reactions with 1-methoxy-4-(methylthio)benzene as follows: (i) a single reaction of compound I (or compound II) with the substrate and (ii) the formation of a complex between compound I or II and the substrate followed by reaction of the productive complex with another molecule of sulfide. The overall rate of reaction path ii is faster than that of path i. The preference for path i or ii is highly dependent upon the concentration of sulfide with step ii favored at higher sulfide concentrations. The experimental results obtained on the overall reaction under both pseudo-first-order and single-turnover conditions indicate that compound II reacts competitively with both the organic sulfide substrate and the sulfur cation radical produced from compound I oxidation of sulfide. 相似文献
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The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH. 相似文献
10.
Using both rapid-scan and conventional spectrophotometry, oxygenation of p-substituted thioanisoles by horseradish peroxidase compounds I and II was investigated at pH 5, 7 and 9. The pH-jump technique was applied to the compound II reactions at acidic and neutral pH. The rate of oxidation of the sulfides is dependent on pH, concentration of substrate and on the different substituents in the para position of the benzene ring. Our results, based on transient state observations of the enzyme intermediates, are in agreement with the results of Kobayashi, S., Minoru, N., Kimura, T. and Schaap, A.P. (Biochemistry (1987) 26, 5019-5022), obtained using 18O-labelling and studies of product formation, in which formation of a sulfur cation radical from compound I is proposed. We consider two reaction mechanisms for the compound II reaction: one a one-electron oxidation of the thioanisole, analogous to the compound I reaction, and the other, the attack of the hydroxyl radical originating from compound II on the sulfur-cation radical. 相似文献
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Optical and magnetic measurements of horseradish peroxidase. I. Azide complex of peroxidase 总被引:1,自引:0,他引:1
M Tamura 《Biochimica et biophysica acta》1971,243(2):239-248
13.
Shintaku M Matsuura K Yoshioka S Takahashi S Ishimori K Morishima I 《The Journal of biological chemistry》2005,280(49):40934-40938
A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1). 相似文献
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W R Browett Z Gasyna M J Stillman 《Biochemical and biophysical research communications》1983,112(2):515-520
The magnetic circular dichroism spectrum of the compound I species of horseradish peroxidase, which contains an iron (IV) porphyrin pi-cation radical complex, has been measured between 273 K and 4.2 K. The spectrum is temperature independent between 273 K and 30 K. However, very strong temperature dependence is observed below 30 K. These data do not appear to fit the temperature dependence expected for the presence of a simple MCD C term, or combination of C terms, but suggest that an increase in the coupling between the S = 1 iron (IV), and the S = 1/2 porphyrin pi-cation radical occurs forming a degenerate ground state. This increase in coupling below 30 K may be the result of a phase change in the protein which in turn affects the electronic structure of the heme group. 相似文献
17.
Compound I of horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase EC 1.11.1.7) was studied by EPR at low temperatures. An asymmetric signal was found, about 15 Gauss wide and with a g-value of 1.995, which could be detected only at temperatures below 20 K and which had an intensity corresponding to about 1% of the heme content. In a titration with H2O2, the signal intensity was proportional to the concentration of Compound I, reaching a maximum when equivalent amounts of H2O2 were added. This indicates that the signal is not due to an impurity, and it is suggested that a free radical is formed, relaxed by a near-by fast-relaxing iron. 相似文献
18.
Rate constants for the reaction between horseradish peroxidase compound I and p-cresol have been determined at several values of pH between 2.98 and 10.81. These rate constants were used to construct a log (rate) versus pH profile from which it is readily seen that the most reactive form of the enzyme is its most basic form within this pH range so that base catalysis is occurring. At the maximum rate a second order rate constant of (5.1 +/- 0.3) x 10(-7) M-1 s-1 at 25 degrees is obtained. The activation energy of the reaction at the maximum rate was determined from an Arrhenius plot to be 5.0 +/- 0.5 kcal/mol. Evidence for an exception to the generally accepted enzymatic cycle of horseradish peroxidase is presented. One-half molar equivalent of p-cresol can convert compound I quantitatively to compound II at high pH, whereas usually this step requires 1 molar equivalent of reductant. The stoichiometry of this reaction is pH-dependent. 相似文献
19.
Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.
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Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation. 相似文献
20.
The rates of reduction of horseradish peroxidase compound II by p-methoxyphenol (4-hydroxyanisole) have been studied from pH 6.0 to 10.5. The kinetics are influenced by an acid group of pKa 8.7 on compound II. The acidic form of compound II is reactive; the basic form is not. Only the electrically neutral, unionized form of p-methoxyphenol is reactive. Fifteen different phenols were reacted with compound II at either pH 7.6 or pH 7.0 (three of them at both pH's). Rate constants varied from zero for p-nitrophenol to 3.2 X 10(7) M-1 for p-aminophenol. The reactive m- and p-substituted phenols yield a rho value of -4.6 +/- 0.5 when plotted according to the Hammett relation. This compares to the rho value of -6.9 obtained for horseradish peroxidase compound I reactions with phenols (1976, D. Job and H. B. Dunford, Eur. J. Biochem. 66, 607). The difference in sensitivity of compounds I and II to electron donating substituents on the phenols can be explained in terms of the relative simplicity of the reactions. Electron donation occurs to the electron-deficient porphyrin pi-cation radical of compound I accompanied by single proton addition to the protein. For compound II the electron is fed to the ferryl group at the center of the porphyrin in a reaction accompanied by two proton additions to the ferryl oxygen atom, one from the protein and the other from the substrate or solvent. This is followed by loss of water from the inner coordination sphere of the ferric ion. The relative reactivities of three o-substituted phenols can be explained in terms of steric hindrance which is minimal for a single o-substituent. 相似文献