The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The role of alkyl chain length in the inhibitory effect n-alkyl xanthates on mushroom tyrosinase activities

Sodium salts of four n-alkyl xanthate compounds, C2H5OCS2Na (I), C3H7OCS2Na (II), C4H9OCS2Na (III), and C6H13OCS2Na (IV) were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) in 10 mM sodium phosphate buffer, pH 6.8, at 293 K using UV spectrophotometry. 4-[(4-Methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed, competitive or uncompetitive inhibition for the four xanthates. For the cresolase activity, I and II showed uncompetitive inhibition but III and IV showed competitive inhibition pattern. For the catecholase activity, I and II showed mixed inhibition but III and IV showed competitive inhibition. The synthesized compounds can be classified as potent inhibitors of MT due to their Ki values of 13.8, 11, 8 and 5 microM for the cresolase activity, and 1.4, 5, 13 and 25 microM for the catecholase activity for I, II, III and IV, respectively. For the catecholase activity both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail of these compounds. The length of the hydrophobic tail of the xanthates has a stronger effect on the Ki values for catecholase inhibition than for cresolase inhibition. Increasing the length of the hydrophobic tail leads to a decrease of the Ki values for cresolase inhibition and an increase of the Ki values for catecholase inhibition.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C₃H₇OCS₂Na (I), C₄H₉OCS₂Na (II) and C₅H₁₁OCS₂Na (III), were synthesized, by the reaction between CS₂ with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K_i values of 9.8, 7.2 and 6.1 μM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S^? ) of the anion xanthate, which leads to similar values of K_i for all three xanthates. Different K_i values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C4H9NHCS2Na (I), C6H13NHCS2Na (II) and C8H17NHCS2Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to Ki values of 0.8, 1.0 and 1.8 microM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different Ki values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30 degrees C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K(i)) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K(i) value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30 degrees C at both pHs. Moreover, the effect of temperature on K(i) value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30°C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K_i) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K_i value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30°C at both pHs. Moreover, the effect of temperature on K_i value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C₄H₉NHCS₂Na (I), C₆H₁₃NHCS₂Na (II) and C₈H₁₇NHCS₂Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver–Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to K_i values of 0.8, 1.0 and 1.8 μM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different K_i values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

The inhibitory effect of ethylenediamine on mushroom tyrosinase

The inhibitory effect of ethylenediamine on both activities of mushroom tyrosinase (MT) at 20 °C in a 10 mM phosphate buffer solution (pH 6.8), was studied. L-DOPA and L-tyrosine were used as substrates of catecholase and cresolase activities, respectively. The results showed that ethylenediamine competitively inhibits both activities of the enzyme with inhibition constants (K(i)) of 0.18±0.05 and 0.14±0.01 μM for catecholase and cresolase respectively, which are lower than the reported values for other MT inhibitors. For further insight a docking study between tyrosinase and ethylenediamine was performed. The docking simulation showed that ethylenediamine binds in the active site of the enzyme near the Cu atoms and makes 3 hydrogen bonds with two histidine residues of active site.     

Differential effect of phenylhydrazine on the catecholase and cresolase activities of Vitis catechol oxidase

The simultaneous addition of phenylhydrazine and p-cresol to grape catechol oxidase resulted in enhanced oxidation of p-cresol. Carbonyl reagents such as hydrazine, borohydride and semicarbazide also enhanced cresolase activity but had no effect on catecholase activity. Pretreatment of the enzyme with periodate abolished cresolase activity. The effects of periodate and ascorbate or semicarbazide on cresolase activity were mutually reversible. The simultaneous addition of phenylhydrazine and 4-methylcatechol to the enzyme did not result in inhibition of the initial rate of oxidation of the phenolic substrate. It is concluded that phenylhydrazine does not react with a carbonyl group on the enzyme. The possible involvement of conformational changes in the enzyme, determining phenylhydrazine inhibition is discussed.     

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.     

Stability, structural and suicide inactivation changes of mushroom tyrosinase after acetylation by N-acetylimidazole

Modification (acetylation) of Tyr residues with N-acetylimidazole protects outstandingly mushroom tyrosinase (MT) from the suicide inactivation in the presence of its catecholic substrate, 4-[(4-methylbenzo) azo]-1,2-benzenediol. UV spectrophotometric experiments and differential scanning calorimetry (DSC) studies indicated a decrease in kinetic stability of the enzyme alongside with increase in its thermal stability as well as its stability against n-dodecyl trimethylammonium bromide as a denaturizing agent. Pace analysis resulted in standard Gibbs free energy values of 46.54 and 52.09 kJ/mol in the absence of denaturant for native and modified enzyme, respectively. Structural studies by circular dichroism (CD) spectrophotometry showed that modification did not have major impact on the secondary structure of MT; however, induced some changes in its tertiary structure. The near-UV CD results revealed that the modification had enhanced intramolecular van der Waals interactions in the enzyme structure, which was in coincidence with its thermodynamic stability.     

Dual effects of aliphatic carboxylic acids on cresolase and catecholase reactions of mushroom tyrosinase

Catecholase and cresolase activities of mushroom tyrosinase (MT) were studied in presence of some n-alkyl carboxylic acid derivatives. Catecholase activity of MT achieved its optimal activity in presence of 1.0, 1.25, 2.0, 2.2 and 3.2?mM of pyruvic acid, acrylic acid, propanoic acid, 2-oxo-butanoic acid, and 2-oxo-octanoic acid, respectively. Contrarily, the cresolase activity of MT was inhibited by all type of the above acids. Propanoic acid caused an uncompetitive mode of inhibition (K_i=0.14?mM), however, the pyruvic, acrylic, 2-oxo-butanoic and 2-oxo-octanoic acids showed a competitive manner of inhibition with the inhibition constants (K_i) of 0.36, 0.6, 3.6 and 4.5?mM, respectively. So, it seems that, there is a physical difference in the docking of mono- and o-diphenols to the tyrosinase active site. This difference could be an essential determinant for the course of the catalytic cycle. Monophenols are proposed to bind only the oxyform of the tyrosinase. It is likely that the binding of acids occurs through their carboxylate group with one copper ion of the binuclear site. Thus, they could completely block the cresolase reaction, by preventing monophenol binding to the enzyme. From an allosteric point of view, n-alkyl acids may be involved in activation of MT catecholase reactions.     

Tyrosinase-like activity and estradiol binding in rat uterine nuclear extracts.

Nuclear extracts from the uteri of estradiol-implanted rats contain a tyrosinase-like enzyme that has three activities: monophenolase or cresolase, diphenolase or catecholase, and estrogen binding. When [3H]estradiol was used as a substrate, 3H2O was released from the A ring in the presence of copper and ascorbic acid. The optimal concentrations of these cofactors for the cresolase activity were established. The cresolase activity was lost on attempts at further purification. Estradiol binding was observed in conjunction with the enzymatic activity and was dependent on the presence of ascorbic acid and copper. The most potent inhibitors of 3H2O release from [3H]estradiol were those with a dihydroxyphenol moiety. The reaction was also sensitive to sulfhydryl reagents. These features of the enzyme are distinctive from other oxidases capable of attacking the aromatic ring of estrogens.     

Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates

Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented.     

Enzymatic oxidation by frog epidermis tyrosinase of 4-methylcatechol and p-cresol. Influence of L-serine

A study has been made of the kinetics of cresolase and catecholase activities of tyrosinase on the p-cresol/4-methyl-catechol pair in the presence of L-serine. For this, a spectrophotometer assay for both activities based on the spectrophotometric and stoichiometric characteristics of the chemical reactions in the evolution of 4-methyl-o-benzoquinone is proposed. The presence of L-serine in the reaction medium caused a modification in the lag period and the steady-state rate expressed during the cresolase activity of tyrosinase, but no modification was observed during the expression of catecholase activity. These results can be explained taking into account the complex mechanism proposed for tyrosinase which included the chemical steps involved in the process. Furthermore, a singular form of regulation of enzymatic activity by L-serine has been clarified, not by any direct interaction between the protein molecule and the nucleophile, but by modification of the chemical reactions involved in the mechanism of tyrosinase.     

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.     

Successful resonance Raman study of cresolase activity of mushroom tyrosinase

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.     

Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.     

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.     

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.