How do enzymes work? Effect of electron circuits on transition state acid dissociation constants.

The effect of electron flow through a complete circuit on transition state acid dissociation constants is used to explain the remarkable catalysis observed in a redox reaction, the formation of compound I from native peroxidase. The explanation for the huge shift in the dissociation constant of a distal histidine residue, in going from the resting enzyme to the transition state, is a complete electron circuit through many amino acid residues and hydrogen bonds which prevents the development of localized charge. The key feature is electron flow through the circuit at the instant that proton transfer is occurring in the opposite direction. Electron flow occurs in one direction for attainment of the transition state and in the opposite direction for product formation.

Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion.

The reaction of myeloperoxidase compound I (MPO-I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 x 106 M-1.s-1 measured at 25 degrees C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem. 270, 30434-30440], and 2.5 x 104 M-1.s-1 at 15 degrees C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry 37, 17923-17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO-I + Cl- --> MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following: MPO+H2O2 ⇄k-1k1 MPO-I+H2O MPO-I+Cl- ⇄k-2k2 MPO-I-Cl- MPO-I-Cl- -->k3 MPO+HOCl where MPO-I-Cl- is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate-controlling at low [Cl-]. At high [Cl-], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl-] is a composite rate constant, approximated by k2k3/k-2. Values of k1 and k-1 are known from the literature. Results of this study yielded k2 = 2.2 x 106 M-1.s-1, k-2 = 1.9 x 105 s-1 and k3 = 5.2 x 104 s-1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.

Peroxidase-catalyzed halide ion oxidation.

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I*, although at a slower rate than oxidation of I by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe-HOCl or X-HOCl where X is an amino acid residue), or molecular chlorine Cl2. A study of the nonenzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I2. It was impossible to tell which species because of the equilibria: [reaction: see text] The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl2 does not prove it is the chlorinating species. If Cl2 is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.

Oxidation of clozapine and ascorbate by myeloperoxidase.

The kinetics and spectra of the reactions of clozapine with compounds I and II of myeloperoxidase were investigated using both single- and sequential-mixing stopped-flow techniques, steady-state kinetics, and spectrophotometric measurements. The results show conclusively that both compounds I and II are reduced in one-electron reactions with clozapine. At pH 7.0 the rate constant for compound I reacting with clozapine is (1.5 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (4.8 +/- 0.1) x 10(4) M(-1) s(-1). The physiological pH of 7.4 was found to be optimal for the oxidation of clozapine by compound I. The rate constant for compound I reacting with ascorbate is (1.1 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (1.1 +/- 0.2) x 10(4) M(-1) s(-1), both obtained at pH 7.0. Experiments with both clozapine and ascorbate present showed that ascorbate acts both as a competitive inhibitor and free radical scavenger.

Reactions of prostaglandin endoperoxide synthase with hydroperoxide and reducing substrates under single turnover conditions.

The peroxidase reaction of prostaglandin endoperoxide synthase was investigated by transient state kinetics using stoichiometric amounts of substrates. The rate constants for the conversion of compound I to intermediate II determined with a stoichiometric amount of hydroperoxide were found to be lower by an order of magnitude than when an excess of hydroperoxide was used. The difference was attributed to ability of the compound I of prostaglandin endoperoxide synthase to be reduced by the excess of hydroperoxide. This suggests that the true rate constant of unimolecular conversion compound I to intermediate II at 3 degrees C is 5-10 s-1 instead of 50-200 s-1 as reported before. The latter value rather characterizes the combined process of spontaneous and hydroperoxide-dependent transformation of compound I. Stoichiometric amounts of reducing substrates significantly stimulated transformation of compound I. This effect could not be entirely explained by their reducing action, which was measured by following the oxidation kinetics. The results of the global fit of the experimental data suggest that reducing substrates, in addition to their direct action in reducing compound I to compound II, indirectly stimulate transformation of compound I to the tyrosyl radical form of intermediate II, thereby stimulating the cyclooxygenase reaction.

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.

The reaction rates of NO with horseradish peroxidase compounds I and II.

In this study the reactions between nitric oxide (NO) and horseradish peroxidase (HRP) compounds I and II were investigated. The reaction between compound I and NO has biphasic kinetics with a clearly dominant initial fast phase and an apparent second-order rate constant of (7.0 +/- 0.3) x 10(5) M(-1) s(-1) for the fast phase. The reaction of compound II and NO was found to have an apparent second-order rate constant of k(app) = (1.3 +/- 0.1) x 10(6) M(-1) s(-1) or (7.4 +/- 0.7) x 10(5) M(-1) s(-1) when measured at 409 nm (the isosbestic point between HRP and HRP-NO) and 419 nm (lambda(max) of compound II and HRP-NO), respectively. Interestingly, the reaction of compound II with NO is unusually high relative to that of compound I, which is usually the much faster reaction. Since horseradish peroxidase is prototypical of mammalian peroxidases with respect to the oxidation of small substrates, these results may have important implications regarding the lifetime and biochemistry of NO in vivo after inflammation where both NO and H(2)O(2) generation are increased several fold.

The proofreading pathway of bacteriophage T4 DNA polymerase.

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.

Reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11,14-dienoic acid.

The pre-steady-state phase of the oxygenase reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11, 14-dienoic acid has been studied using stopped flow techniques. Because some intermediate forms of prostaglandin endoperoxide synthase are spectrally indistinguishable, the enzyme and substrate transformations were monitored in parallel to simplify the interpretation of the kinetics. Over a wide range of conditions, the formation of the enzyme intermediate II, the form of compound I containing the tyrosyl radical, precedes substrate oxidation. This result supports the occurrence of a unimolecular conversion of compound I into intermediate II. Furthermore, the rate of intermediate II formation was stimulated by increased concentration of dienoic acid, perhaps because of increased occupation of the fatty acid binding site. The importance of the unimolecular formation of intermediate II was confirmed by simulated kinetics of the oxygenase reaction. These results provide evidence that intermediate II is the primary oxidant in the reaction of prostaglandin synthase with the dienoic acid, as it is with arachidonic acid.

Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics.

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.

pH dependence and structural interpretation of the reactions of Coprinus cinereus peroxidase with hydrogen peroxide, ferulic acid, and 2,2'-azinobis.

Steady-state and transient-state analysis of Coprinus cinereus peroxidase, CIP (identical to Arthromyces ramosus peroxidase), was used to characterize the kinetics of the three fundamental steps in heme peroxidase catalysis: compound I (cpd I) formation, cpd I reduction, and compound II (cpd II) reduction. The rate constant k1 for cpd I formation determined by transient-state analysis is (9.9 +/- 0.6) x 10(6) M-1 s-1. The k1 determined by steady-state analysis is (8.8 +/- 0.6) x 10(6) M-1 s-1 in the presence of ferulic acid and (6.7 +/- 0.2) x 10(6) M-1 s-1 in the presence of ABTS. The value of k1 is constant from pH 6 to 11. However, at low pH the value of k1 decreases, corresponding to titration of an enzyme group with a pKa of 5.0. Titration of this group is also detected from cyanide-binding kinetics. Furthermore, titration of this group is linked with marked spectroscopic changes unique to CIP. We ascribe these changes to protonation of proximal His183. A very low pKa is proposed for distal His55 in the resting state of CIP. The rate constants, k2 for cpd I and k3 for cpd II reduction, are very large for both ferulic acid and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). For ferulic acid, transient-state kinetic analysis shows that the values of k2 and k3 are identical at pH 5-6, and the ratio k2/k3 increases to 10 at pH 10. The similar magnitude of k2 and k3 is unusual for a peroxidase. Both k2 and k3 decrease with increasing pH, and both are influenced by two ionizations: one with a pKa value near 7, assumed to reflect the protonation of His55; and the other with pKa of 9.0 +/- 0.7 for k2 and 8.8 +/- 0.4 for k3, perhaps reflecting the phenol-linked deprotonation of ferulic acid. Steady-state analysis at pH 7.0 gave k2k3/(k2 + k3) = (2.2 +/- 0.1) x 10(7) M-1 s-1 for ferulic acid, and (2.0 +/- 0.7) x 10(7) M-1 s-1 for ABTS and revealed a unimolecular step with ku = 1500 s-1, ascribed to slow ABTS radical product release. From transient-state results at pH 7, the values of k2 and k3 were found to be identical also for ABTS. A mechanism for cpd I and II reduction involving distal histidine and arginine is proposed.

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase.

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with H2O2 is 4.O x 10(7) M-1 s-1, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(IV)-porphyrin-pi-cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for compound I reduction by ascorbate is extremely fast (8.0 x 10(7) M-1 S-1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pH-rate profile for compound I formation indicates a pKa value of 5.0 for a group affecting the active site reaction.

Effect of Trolox C on the oxygenation reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11, 14-dienoic acid.

Trolox C, a water-soluble derivative of alpha-tocopherol, stimulates the oxygenation of cis,cis-eicosa-11, 14-dienoic acid (AH) by prostaglandin endoperoxide synthase at lower concentrations and suppresses the stimulated reaction at higher concentrations. Surprisingly, Trolox C does not affect the stoichiometric ratio between the rate of formation of the oxygenation product 11-hydroxy-12-trans, 14-cis-eicosadienoic acid (AOH) and the rate of disappearance of molecular oxygen. The ratio of the two rates, d[AOH]/-d[O2], remains constant at 2/1 for a series of Trolox C concentrations and in the absence of Trolox C. Results indicate that AH reacts preferentially with Compound I of the enzyme and that Trolox C does not compete for Compound I. Enzyme inactivation begins with formation of an unproductive Compound I-tyrosyl radical (Compound I-X.) which has the same number of oxidizing equivalents as the conventional peroxidase Compound I. The stimulating effect of low concentrations of Trolox C can be explained by reduction of the oxyferryl heme so that Compound I-X. is reduced to a Compound II-X.species, the Compound II analog of Compound I-X.. Thus heme bleaching is prevented. A further one-electron reduction by Trolox C of Compound II-X. reforms the native enzyme, which permits enzyme recycling. Large concentrations of Trolox C inhibit reformation of native enzyme, reducing the extent of stimulation.

Reactions of prostaglandin endoperoxide synthase and its compound I with hydroperoxides.

The reactions of native prostaglandin endoperoxide synthase with structurally different hydroperoxides have been investigated by using kinetic spectrophotometric scan and conventional and sequential mixing stopped-flow experiments. The second order rate constants for compound I formation are (5.9 +/- 0.1) x 10(4) M-1 s-1 using t-butyl hydroperoxide as the oxidant, (2.5 +/- 0.1) x 10(6) M-1 s-1 for ethyl hydroperoxide and (5.1 +/- 0.6) x 10(7) M-1 s-1 for m-chloroperoxybenzoic acid at pH 7.0, 6.7 +/- 0.2 degrees C, and ionic strength 0.1 M. Sequential mixing, transient state experiments show for the first time that all hydroperoxides reduce compound I in a bimolecular reaction. Ethyl hydroperoxide, t-butyl hydroperoxide, and m-chloroperoxybenzoic acid react directly with compound I. The natural substrate prostaglandin G2 forms a transient complex with compound I before the reduction step occurs. Therefore, compound I initially transforms to compound II, not to the compound I-tyrosyl radical. Second order rate constants for the reactions of compound I are (2.9 +/- 0.2) x 10(4) for t-butyl hydroperoxide, (3.5 +/- 0.5) x 10(4) for hydrogen peroxide, (4.2 +/- 0.2) x 10(4) for ethyl hydroperoxide, and (4.2 +/- 0.3) x 10(5) for m-chloroperoxybenzoic acid, all in units of M-1 s-1 and same conditions as for compound I formation. The rate of reaction of prostaglandin G2 with compound I, calculated from the ratio of kcat to Km obtained from the saturation curve, is (1.0 +/- 0.2) x 10(6) M-1 s-1 at 3.0 +/- 0.2 degrees C. Results are discussed in the context of the current state of knowledge of the mechanisms of the cyclooxygenase and peroxidase reactions of prostaglandin endoperoxide synthase.

pH and temperature dependence of the rate of compound I formation from the reaction of prostaglandin endoperoxide synthase with hydrogen peroxide.

The formation of primary oxidized compound of prostaglandin endoperoxide synthase, compound I, was studied as a function of pH and temperature using hydrogen peroxide as a substrate. Analysis of the results indicates that compound I formation is influenced by an ionizable group with a pKa of 4.06 +/- 0.04. The protonated form of hydrogen peroxide preferentially reacts with the unprotonated form of the enzyme over the pH range of 3.5 to 9.1, suggesting the importance of acid-base catalysis for compound I formation. The second-order rate constant for the reaction of the enzyme with hydrogen peroxide in the pH-independent region is (4.6 +/- 0.2) x 10(5) M-1 S-1 at an ionic strength of 0.1 M and temperature of 4.0 +/- 0.2 degrees C. The effect of temperature on the rate of compound I formation was studied from 3.4 to 24.1 degrees C in the pH-independent region (pH 6.98) and at a constant ionic strength of 0.1 M. The kinetic parameters obtained from the temperature dependence are the following: Arrhenius activation energy, Ea = 102 +/- 5 kJ/mol; free energy of activation, delta G++, 36 +/- 3 kJ/mol; enthalpy of activation, delta H++, 100 +/- 5 kJ/mol; entropy of activation, delta S++, 215 +/- 9 J/mol K. These activation values are very different from those obtained for the reactions of other peroxidases and catalases with hydrogen peroxide, indicating profound differences in active site structure.

Oxygenation reactions of prostaglandins endoperoxide synthase and soybean lipoxygenase. Surprising stoichiometry in the formation of hydroperoxy and hydroxy derivatives of cis,cis-eicosa-11,14-dienoic acid.

The stoichiometry of the oxygenation reaction of cis,cis-eicosa-11,14-dienoic acid catalyzed by prostaglandin endoperoxide synthase and soybean lipoxygenase has been investigated by using steady-state initial rate measurements. The rate of product formation (conjugated diene hydroperoxy and hydroxy derivatives) was followed spectrophotometrically at 235 nm, and the rate of oxygen consumption was measured polarographically. The ratio of the two rates, d[conjugated diene/-d[O2], is 2/1 for the prostaglandin endoperoxide synthase catalyzed reaction and 1/1 for the lipoxygenase reaction. The 2/1 ratio can be explained by two interrelated routes, each of which results in formation of the conjugated diene hydroxy derivative of the acid. One route, initiated by hydrogen atom abstraction from the acid by Compound I, results in formation of the conjugated diene hydroperoxy derivative. The latter is converted to the hydroxy derivative by regenerating Compound I from the native enzyme. The other route involves direct oxygen atom insertion into the acid by the tyrosyl radical form of Compound I. The decrease in absorbance at 235 nm obtained in the presteady-state phase suggests that during the initial contact of hydroperoxide and enzyme an epoxy-hydroxy fatty acid-enzyme complex may be formed.

Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies.

The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.

Transient and steady-state kinetics of the oxidation of scopoletin by horseradish peroxidase compounds I, II and III in the presence of NADH.

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxidase. However, the kinetics of the elementary steps of the reaction have never been investigated, nor has the quantitative effect of interfering substances ever been explored in detail, despite the fact that scopoletin is widely used in a peroxidase assay for H2O2. In this work, we employed both transient-state and steady-state methods to determine the second-order rate constants for the oxidation of scopoletin by the horseradish peroxidase (HRP) intermediate compounds I and II: (3.7 +/- 0.1) x 10(6) M-1 s-1 and (8.5 +/- 0.5) x 10(5) M-1 s-1 at 20 degrees C, pH 6.0 and ionic strength of 0.1 M. We investigated the possible inhibitory effect of NADH on the reaction of scopoletin with HRP and also the effect of scopoletin on the NADH reaction. In the presence of NADH the rate constant for the reaction between HRP-I and scopoletin decreased slightly to (2.8 +/- 0.1) x 10(6) M-1 s-1. Thus, although NADH is also a peroxidase substrate, it cannot compete effectively for the oxidized forms of the enzyme. On the other hand, scopoletin stimulates the oxidation of NADH by the HRP/H2O2 system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD. radicals. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added H2O2) HRP-II is the dominant enzyme intermediate with HRP-III also detectable. Addition of scopoletin to the HRP/NADH system leads to a biphasic reaction in which HRP-II and HRP-III disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribute the faster phase to the HRP-II reaction with scopoletin with a rate constant of (6.2 +/- 0.1) x 10(5) M-1 s-1 and the slower phase to the HRP-III reaction with scopoletin with rate constant (5.0 +/- 0.4) x 10(4) M-1 s-1. Our present work not only provides rate constants for the oxidation of scopoletin by HRP-I, II and III but also elucidates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.

One-electron oxidations by peroxidases.

1. Peroxidases typically follow the reaction cycle: native enzyme-->compound I-->compound II-->native enzyme, in which the latter two steps involve hydrogen atom transfer from substrate to enzyme. 2. Exceptions involve (1) very facile, rapidly reacting reducing substrates that transfer an electron rather than a hydrogen atom, resulting in formation of a substrate pi-cation radical; (2) two two-electron transfer steps: native enzyme-->compound I-->native enzyme; and (3) compound III and the reduced form of the enzyme containing iron(II). 3. Prostaglandin H synthase is a peroxidase with some of the properties of a P450 in that compound I can abstract the hydrogen atom from a C-H bond. 4. The so-called cyclooxygenase and peroxidase activities of prostaglandin H synthase are intimately connected and, with the above exception, both are part of a conventional peroxidase cycle.