Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.     

Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators.

Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.     

Mechanism of cytochrome c peroxidase. O-benzoylhydroxylamine as an analog of hydrogen peroxide.

A number of reagents, some of which are electronic analogs of hydrogen peroxide, will replace it in the reactions of cytochrome c peroxidase. These compounds include N-bromosuccinimide, sodium hypochlorite, and the novel oxidizing agent O-benzoylhydroxylamine. If fragments of the oxidant played a functional role in the structure of the oxidized form of the enzyme, it would be expected that the product formed from O-benzoylhydroxylamine would differ from that formed from hydrogen peroxide. The products formed on reaction of the two oxidizing agents with cytochrome c peroxidase are indistinguishable. This results carries implications for the structure of the so-called ES compound. The extension in the range of specific substrates for cytochrome c peroxidase allows identification of the structure which compounds must possess to be oxidizing substrates for the enzyme. A mechanism for the first step of the reaction is suggested. O-Benzoylhydroxylamine is also a reducing agent, and its reaction with the enzyme is analogous to that of hydrogen peroxide with catalase. The final product of the reaction is the inert nitric oxide complex of ferrous cytochrome c peroxidase.     

Analysis of the steady-state mechanism of the aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase from yeast.

The steady-state mechanism of the aminoacylation of tRNAPhe by the corresponding synthetase from yeast has been investigated in detail by kinetic experiments. It was found that there are two alternative mechanisms: one favoured at low tRNA concentrations and the other at high tRNA concentrations. ATP and Phe are bound randomly to the enzyme. AMP is released immediately after the binding of ATP and Phe. Between the release of AMP and pyrophosphate (PPi) there is at least one additional step. Based on the experimental results a model of the steady-state mechanism is proposed. This model includes the sequence of addition of substrates to the enzyme and the release of products from the enzyme as well as the composition of the intermediate complexes with the enzyme. This model is in accordance with previous results based on different techniques. The results are explained by a "flip-flop" mechanism for all the substrates and products involved in the reaction.     

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.     

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Role of oxygen during horseradish peroxidase turnover and inactivation

Horseradish peroxidase catalyzed oxidation of phenol has been reinvestigated to determine the requirements of facile enzyme autoinactivation. Turnover of this peroxidase was monitored spectrophotometrically at 400 nm and found dependent on the concentration of phenol and hydrogen peroxide. The inactivation of the peroxidase required both substrates, phenol and H2O2, but surprisingly was also potentiated by molecular oxygen. Exclusion of diffusible superoxide or hydroxyl radicals had slight effect on product formation or loss of catalytic activity. A mechanism is proposed to explain the unanticipated role of oxygen during enzyme inactivation.     

Peptidylglycine-alpha-hydroxylating monooxygenase generates two hydroxylated products from its mechanism-based suicide substrate, 4-phenyl-3-butenoic acid

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.     

Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the front-line antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of M. tuberculosis NAT which reveals that substituted anilines are excellent substrates but that isoniazid is a very poor substrate for this enzyme. We propose that the expression of NAT from M. tuberculosis (TBNAT) is unlikely to be a significant cause of isoniazid resistance. The kinetic parameters for a variety of TBNAT substrates were examined, including 3-amino-4-hydroxybenzoic acid and AcCoA, revealing K m values of 0.32 +/- 0.03 and 0.14 +/- 0.02 mM, respectively. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a bi-bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters reveals that one enzyme group must be deprotonated for optimal catalytic activity and that two amino acid residues at the active site of the free enzyme are involved in binding and/or catalysis. Solvent kinetic isotope effects suggest that proton transfer steps are not rate-limiting in the overall reaction for substituted aniline substrates but become rate-limiting when poor hydrazide substrates are used.     

Coupling of the peroxidase and cyclooxygenase reactions of prostaglandin H synthase

Interrelations between peroxidase and cyclooxygenase reactions catalyzed by prostaglandin endoperoxide synthase (prostaglandin H synthase) were analyzed in terms of the mutual influence of these reactions. The original branched-chain mechanism predicts competition between these two reactions for enzyme, so that peroxidase cosubstrate should inhibit the cyclooxygenase reaction and the cyclooxygenase substrate is expected to inhibit the peroxidase reaction. In stark contrast, the peroxidase reducing substrate is well known to strongly stimulate the cyclooxygenase reaction. In the present work the opposite effect, the influence of the cyclooxygenase substrate on the peroxidase reaction was studied. Experiments were conducted on the effect of arachidonic acid on the consumption of p-coumaric acid by prostaglandin H synthase and 5-phenyl-4-pentenyl-1-hydroperoxide. Neither the steady-state rates nor the total extent of p-coumaric acid consumption was affected by the addition of arachidonic acid. This suggests that the cyclooxygenase substrate does not influence observable velocities of the peroxidase reaction, namely oxidation and regeneration of the resting enzyme. The data support coupling of the cyclooxygenase and peroxidase reactions. A combination of the branched-chain and tightly coupled mechanisms is proposed, which includes a tyrosyl radical active enzyme intermediate regenerated through the peroxidase cycle. Numerical integration of the proposed reaction scheme agrees with the observed relations between peroxidase and cyclooxygenase reactions in the steady state.     

Some aspects of the origin and early evolution of bioenergetic processes

Model experiments demonstrating some possibilities of the primitive bioenergetics were performed. Comparing the catalytic properties of heme and hemoproteinoid in electron transfer reactions, it was shown that hemin acts as an active dark catalyst, whereas hemoproteinoid is a photosensitizer of these processes. The heme-containing enzyme, peroxidase/or even the hemoproteinoid/is also able to provide the phosphorylation of different substrates. It is proposed, that the phosphorylation is initiated by a hydroxyl radical occuring in the peroxidase reaction. We believe that those reactions could play a role in prebiotic energetics by coupling the electron transfer with the phosphorylation of suitable substrates.     

Some aspects of the origin and early evolution of bioenergetic processes

Model experiments demonstrating some possibilities of the primitive bioenergetics were performed. Comparing the catalytic properties of heme and hemoproteinoid in electron transfer reactions, it was shown that hemin acts as an active dark catalyst, whereas hemoproteinoid is a photosensitizer of these processes. The heme-containing enzyme, peroxidase [or even the hemoproteinoid] is also able to provide the phosphorylation of different substrates. It is proposed, that the phosphorylation is initiated by a hydroxyl radical occuring in the peroxidase reaction. We believe that those reactions could play a role in prebiotic energetics by coupling the electron transfer with the phosphorylation of suitable substrates.     

Hydroperoxyfatty acids inactivate the reticulocyte lipoxygenase independently of a hydroperoxidase reaction

From a comparison of 9Ds-HPODE and 13Ls-HPODE and their methyl esters as substrates and inactivating agents of reticulocyte lipoxygenase it is concluded that the compounds inactivate the enzyme independently of any hydroperoxidase reaction. The protective effect of 4-nitrocatechol indicates the formation of Fe(III) complexes of the enzyme with the hydroperoxyfatty acid compounds prior to inactivation.     

A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.     

An iso-random Bi Bi mechanism for adenylate kinase.

An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.     

Inhibition and inactivation of horseradish peroxidase by thiourea

The kinetics of horseradish peroxidase (EC 1.11.1.7)-catalyzed oxidation of o-dianisidine by hydrogen peroxide in the presence of thiourea were studied. At the first, fast step of this process thiourea acts as a competitive reversible inhibitor with respect to o-dianisidine (Ki = 0.22 mM). The formation of a thiourea-peroxidase complex was determined by the increase in the absorbance at A495 and A638 of the enzyme. The dissociation constant for the peroxidase-thiourea complex is equal to 2.0-2.7 mM. Thiourea is not a specific substrate of peroxidase during the oxidation reaction by H2O2, but is an oxidase substrate (although not a very active one) of peroxidase. The irreversible inactivation of the enzyme during its incubation with thiourea was studied. The first-order inactivation rate constant (kin) was shown to increase with a fall in the enzyme concentration. The curve of the dependence of kin on the initial concentration of thiourea shows a maximum at 5-7 mM. The enzyme inactivation is due to its modification by intermediate free radical products of thiourea oxidation. The inhibitors of the free radical reactions (o-dianisidine) protect the enzyme against inactivation. The degree of inactivation depends on concentrations and ratio of thiourea and peroxidase. A possible mechanism of peroxidase interaction with thiourea is discussed.     

The mechanism of the hypolipidemic effect of garlic oil extract in rats fed on high sucrose and alcohol diets

The effect of feeding garlic oil to white albino rats maintained on high sucrose and alcohol diets was studied. It is proposed that the mechanism of the hypolipidemic effect of the oil involves the active principle, diallyl disulphide, inactivating enzymes and substrates containing thiol groups in an exchange reaction; increased hydrolysis of triacylglycerols as increased lipase activity is induced by the oil; and the reduction in the biosynthesis of triacylglycerols as NADPH is made unavailable for the process by the metabolism of the oil.     

The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.