Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

Superoxide enhances hypochlorous acid production by stimulated human neutrophils

Stimulated neutrophils undergo a respiratory burst discharging large quantities of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and Cl- to hypochlorous acid. Human neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in Cl(-)-free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalase, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorbol myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.     

Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.

Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase.     

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone.

Myeloperoxidase (MPO) is a prime candidate for mediating the inflammatory tissue damage of neutrophils because it converts Cl- to the potent oxidant hypochlorous acid. It also oxidizes xenobiotics to reactive free radicals. We have found that the kinetics of oxidation of hydroquinone by myeloperoxidase are inadequately explained by the classical peroxidase mechanism. Peroxidation of hydroquinone displayed a distinct lag phase, which was practically abolished by excluding O2 and was eliminated by adding benzoquinone at the start of the reaction. Superoxide dismutase increased the rate of peroxidation by 40% but did not eliminate the lag phase. Spectral investigations revealed that during the initial phase of the reaction, MPO was converted to oxy-MPO, or compound III, by a mechanism that was not reliant on superoxide. Benzosemiquinone, however, was able to convert ferric-MPO to compound III. Both compound III and ferro-MPO reacted with benzoquinone to regenerate ferric-MPO. We propose that the lag phase occurs because benzosemiquinone reduces ferric-MPO to ferro-MPO, which rapidly binds O2 to form compound III. Since compound III is outside the peroxidation cycle, conversion of hydroquinone to benzoquinone is retarded. However, as benzoquinone accumulates, it oxidizes ferro-MPO and compound III to ferric-MPO, thereby increasing the rate of peroxidation. There is a minimal lag phase under an atmosphere of N2 because ferro-MPO would be rapidly oxidized by benzoquinone, without formation of compound III. We conclude that when substrates produce radicals capable of reducing ferric-MPO, they will be peroxidized efficiently only if oxy-MPO is readily recycled. Furthermore, these radicals will prevent MP3+ from reacting with H2O2, and thereby prevent the enzyme from oxidizing Cl- to hypochlorous acid. Thus, this mechanism could be exploited to prevent hypochlorous acid-mediated inflammatory tissue damage.     

Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.     

Reaction of myeloperoxidase with its product HOCl.

The reaction of human myeloperoxidase with its product, hypochlorous acid was investigated using both rapid-scan spectrophotometry and the stopped-flow technique. In the reaction of myeloperoxidase with hypochlorous acid a primary compound is found with properties similar to that of compound I and which is converted into compound II. The primary reaction is strongly pH-dependent. At pH 7.2 the reaction is too fast to be measured but at higher pH values it is possible to determine the apparent second-order rate constant. Its value decreases to about 2 x 10(7) M-1.s-1 at pH 8.3 and to 2.3 (+/- 0.4) x 10(6) M-1.s-1 at pH 9.2, respectively. The dissociation constant for the formation of the primary compound is 25.7 (+/- 15.3) microM at pH 9.2 and about 2.5 microM at pH 8.3. The apparent second-order rate constant for the formation of compound II is hardly affected by pH and varies between 2 to 5 x 10(4) M-1.s-1 at pH 10.2 and pH 8.3, respectively. Reaction of myeloperoxidase with hypochlorous acid also resulted in irreversible partial bleaching of the chromophore. Chloride, which is a substrate of the enzyme not only protects myeloperoxidase against bleaching by hypochlorous acid but also competitively inhibits the binding of hypochlorous acid to myeloperoxidase, a process which also has been observed in the reaction with hydrogen peroxide. It is concluded that hypochlorous acid binds at the heme iron to form compound I.     

Differential effects of oxidizing agents on human plasma alpha 1-proteinase inhibitor and human neutrophil myeloperoxidase

Human alpha 1-proteinase inhibitor is easily susceptible to inactivation because of the presence of a methionyl residue at its reactive site. Thus, oxidizing species derived from the myeloperoxidase system (enzyme, H2O2, and C1-), as well as hypochlorous acid, can inactivate this inhibitor, although H2O2 alone has no effect. Butylated hydroxytoluene, a radical scavenger, partially protects alpha 1-proteinase inhibitor from the myeloperoxidase system and completely protects it from hypochlorous acid. Each oxidant also reacts differently with the inhibitor, in that the myeloperoxidase system and hypochlorous acid can each oxidize as many as six methionyl residues, but hypochlorous acid can also oxidize a single tyrosine residue. Myeloperoxidase can be inactivated by hypochlorous acid, by autoxidation in the presence of H2O2 and C1-, as well as by H2O2 alone. Butylated hydroxytoluene completely protects this enzyme from hypochlorous acid inactivation, does not affect the action of H2O2, and enhances autoinactivation. As many as six methionyl residues and two tyrosine residues could be oxidized during autoxidation and six methionine residues by H2O2 alone. Eight methionine residues and one tyrosine residue could be oxidized by hypochlorous acid. The tyrosine residue in myeloperoxidase was oxidized only at a relatively high concentration (600 microM) of hypochlorous acid at which point the enzyme simultaneously and completely lost its enzymatic activity. Loss of activity of myeloperoxidase could also be correlated with the loss of the heme groups present in the enzyme when a relatively high concentration of hypochlorous acid (600 microM) was used and also during autoxidation. It appears that once there is sufficient oxidant to modify one of the tyrosine residues, the heme group itself becomes susceptible.     

Inhibition of myeloperoxidase by salicylhydroxamic acid.

Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events.     

Reaction of human myeloperoxidase with hydrogen peroxide and its true catalase activity

The instability of human myeloperoxidase [EC 1.11.1.7] compound I, which was spontaneously reduced to compound II, and the abnormal stoichiometry of the reaction of myeloperoxidase with H2O2 were investigated. As to the former, a pretreatment of myeloperoxidase with H2O2 did not stabilize compound I, and no difference in its stability was observed between native (alpha 2 beta 2) and hemi (alpha beta) myeloperoxidase. From these results, it was thought that the instability of compound I was caused by neither the presence of endogenous donors nor the intramolecular reduction of compound I to compound II by the other heme in the native enzyme molecule. As for the latter, true catalase activity of myeloperoxidase was demonstrated by monitoring O2 evolution after the injection of H2O2 into the enzyme solution. Myeloperoxidase compound I reacted with H2O2 and returned to the ferric state with concomitant evolution of an O2 molecule. Accordingly, the abnormal stoichiometry of the reaction with H2O2 and a part of the instability of compound I can probably be ascribed to this true catalase activity.     

The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.     

Oxidation of chloride and thiocyanate by isolated leukocytes

Peroxidase-catalyzed oxidation of chloride (Cl-) and thiocyanate (SCN-) was studied using neutrophils from human blood and eosinophils and macrophages from rat peritoneal exudates. The aims were to determine whether Cl- or SCN- is preferentially oxidized and whether leukocytes oxidize SCN- to the antimicrobial oxidizing agent hypothiocyanite (OSCN-). Stimulated neutrophils produced H2O2 and secreted myeloperoxidase. Under conditions similar to those in plasma (0.14 M Cl-, 0.02-0.12 mM SCN-), myeloperoxidase catalyzed the oxidation of Cl- to hypochlorous acid (HOCl), which reacted with ammonia and amines to yield chloramines. HOCl and chloramines reacted with SCN- to yield products without oxidizing activity, so that high SCN- blocked accumulation of chloramines in the extracellular medium. Under conditions similar to those in saliva and the surface of the oral mucosa (20 mM Cl-, 0.1-3 mM SCN-), myeloperoxidase catalyzed the oxidation of SCN- to OSCN-, which accumulated in the medium to concentrations of up to 40-70 microM. Sulfonamide compounds increased the yield of stable oxidants to 0.2-0.3 mM by reacting with OSCN- to yield derivatives analogous to chloramines. Stimulated eosinophils produced H2O2 and secreted eosinophil peroxidase, which catalyzed the oxidation of SCN- to OSCN- regardless of Cl- concentration. Stimulated macrophages produced H2O2 but had low peroxidase activity. OSCN- was produced when SCN- was 0.1 mM or higher and myeloperoxidase, eosinophil peroxidase, or lactoperoxidase was added. The results indicate that SCN- rather than Cl- may be the physiologic substrate (electron donor) for eosinophil peroxidase and that OSCN- may contribute to leukocyte antimicrobial activity under conditions that favor oxidation of SCN- rather than Cl-.     

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.     

Myeloperoxidase inactivation in the course of catalysis of chlorination of taurine.

Myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated from leukocytes of patients with chronic granulocyte leukemia. In the presence of H2O2 and Cl- at pH 4.0-6.6 the myeloperoxidase catalyses chlorination of taurine to monochloramine taurine and simultaneously undergoes inactivation. The myeloperoxidase inactivation rate depends on the concentration of H2O2 and Cl-: both the initial rate of chlorination and myeloperoxidase inactivation rate increase with increasing concentration of H2O2. However, an increase in concentration of Cl- results in a decrease in enzyme inactivation. At a given H2O2 concentration, myeloperoxidase inactivation is a first order reaction, which implied that the enzyme may react with a substrate a limited number of times.     

Reactions of superoxide with myeloperoxidase

When neutrophils ingest bacteria, they discharge superoxide and myeloperoxidase into phagosomes. Both are essential for killing of the phagocytosed micro-organisms. It is generally accepted that superoxide is a precursor of hydrogen peroxide which myeloperoxidase uses to oxidize chloride to hypochlorous acid. Previously, we demonstrated that superoxide modulates the chlorination activity of myeloperoxidase by reacting with its ferric and compound II redox states. In this investigation we used pulse radiolysis to determine kinetic parameters of superoxide reacting with redox forms of myeloperoxidase and used these data in a steady-state kinetic analysis. We provide evidence that superoxide reacts with compound I and compound III. Our estimates of the rate constants for the reaction of superoxide with compound I, compound II, and compound III are 5 x 10(6) M-1 s-1, 5.5 +/- 0.4 x 10(6) M-1 s-1, and 1.3 +/- 0.2 x 10(5) M-1 s-1, respectively. These reactions define new activities for myeloperoxidase. It will act as a superoxide dismutase when superoxide reacts consecutively with ferric myeloperoxidase and compound III. It will also act as a superoxidase by using hydrogen peroxide to oxidize superoxide via compound I and compound II. The favorable kinetics of these reactions indicate that, within the confines of a phagosome, superoxide will react with myeloperoxidase and affect the reactions it will catalyze. These interactions of superoxide and myeloperoxidase will have a major influence on the way neutrophils use oxygen to kill bacteria. Consequently, superoxide should be viewed as a cosubstrate that myeloperoxidase uses to elicit bacterial killing.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons

The effect of H2O2, Cl-, and pH on human myeloperoxidase activity has been examined. The Km for H2O2 is shown to be affected by the combined presence of Cl- and acid pH conditions. The Km for H2O2 is independent of pH in the absence of Cl- and dependent on pH in the presence of Cl-. Conversely, the dependence of the Km for H2O2 on Cl- concentration increases as the pH decreases. A model is proposed in which Cl- has a dual role, acting both as a substrate and as an inhibitor. According to this model, the inhibitor Cl- binding site must be protonated prior to the binding of Cl- and is distinct from the substrate Cl- binding site which is unaffected by pH. The rate equation derived from this model is used to further analyze the data presented. The values of Km for H2O2 predicted by the rate equation are in good agreement with the experimentally determined values.