Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions: IMPLICATIONS FOR THE INHIBITION OF DIOXYGENASE ACTIVITY BY HYDROGEN PEROXIDE*

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H₂O₂) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H₂O₂ inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H₂O₂ consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H₂O₂ consumption and attenuated H₂O₂-induced protein oxidation and dioxygenase inhibition. In the presence of H₂O₂, rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H₂O₂ consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H₂O₂ and NO are elevated and l-Trp is low.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.     

Inactivation of Copper,Zinc superoxide dismutase by H2O2 : Mechanism of protection

Cu,Zn SOD is known to be inactivated by HO₂⁻ and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO₂⁻. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.     

Peroxidase-catalyzed halide ion oxidation

Abstract

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 non-enzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I₂. It was impossible to tell which species because of the equilibria:

I₂+H₂O=HOI+I^-+H⁺</ p>

I^-+I₂=I₃^-

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.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase

The role of active site histidine residues in SCN^– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a second order rate constant of 0.26 M^–1 sec^–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6 indicating histidine derivatization. The UV difference spectrum of the modified versus native enzyme shows a peak at 242 nm indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with SCN^– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically detectable compound II formation is lost due to modification and is not evident after SCN^– protection. The data indicate that out of two histidines, one regulates compound I formation while the other one controls SCN^– binding. SCN^– protected enzyme is inactive due to loss of compound I formation. SCN^– binding studies by optical difference spectroscopy indicate that while the native enzyme binds SCN^– with the K_d of 15 mM, the modified enzyme shows very weak binding with the K_d of 660 mM. From the pH dependent binding of SCN^–, a plot of log K_d vs. pH shows a sigmoidal curve from which the involvement of an enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN^– binding. LGP has thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls SCN^– binding.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

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.     

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular thiol molecule which is considered as a strong non-enzymatic antioxidant. Glutathione regulates multiple metabolic functions; for example, it protects membranes by maintaining the reduced state of both α-tocopherol and zeaxanthin, it prevents the oxidative denaturation of proteins under stress conditions by protecting their thiol groups, and it serves as a substrate for both glutathione peroxidase and glutathione S-transferase. By acting as a precursor of phytochelatins, GSH helps in the chelating of toxic metals/metalloids which are then transported and sequestered in the vacuole. The glyoxalase pathway (consisting of glyoxalase I and glyoxalase II enzymes) for detoxification of methylglyoxal, a cytotoxic molecule, also requires GSH in the first reaction step. For these reasons, much attention has recently been directed to elucidation of the role of this molecule in conferring tolerance to abiotic stress. Recently, this molecule has drawn much attention because of its interaction with other signaling molecules and phytohormones. In this review, we have discussed the recent progress in GSH biosynthesis, metabolism and its role in abiotic stress tolerance.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.     

Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl⁻ restores protection to the original value. The quaternary MgADP–creatine–enzyme–nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V_max. and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.     

Reactivity and specificity of alpha-dicarbonyl compounds towards essential aminoacyl residues of D-beta-hydroxybutyrate dehydrogenase from the inner mitochondrial membrane

The effects of a alpha-dicarbonyl chromophoric reagent: 4-hydroxy-3-nitrophenylglyoxal on the D-beta-hydroxybutyrate dehydrogenase have been compared to those of phenylglyoxal, a specific arginyl reagent in proteins. Both reagents inactivate irreversibly the enzyme. Kinetic experiments show that only one molecule of these reagents per molecule of enzyme is sufficient to inactivate the enzyme. The second order inactivation rate constant is more than 500 times higher with the chromophoric reagent than with phenylglyoxal. A pseudosubstrate (methylmalonate) in presence of coenzyme (NAD) strongly protects enzyme against inactivation by both reagents. Coenzyme alone has no effect on inactivation by phenylglyoxal while it protects whether inhibitor is the chromophoric reagent or N-ethylmaleimide: a thiol specific reagent. These results indicate: 1. That one arginyl residue is essential for D-beta-hydroxybutyrate dehydrogenase activity (experiments with phenylglyoxal). 2. That the presence of a nitro group on position 3 and a hydroxyl-group on position 4 strongly increase the reactivity of the alpha-dicarbonyl groups, but the specificity of the chemical reaction with arginyl residues seems to be lost for the benefit of cysteyl residues.     

The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system

Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes. This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system. When thiol is replaced with another electron donor (e.g. ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation. Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns. The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures. The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities. Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm² were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0°C and characterized by effective first-order rate constants of US inactivation k _in (us) in min^–1. Values of k _in (us) depend on the specific ultrasonic power within the range of 20-60 W/cm², on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO^· radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0°C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm² on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.     

An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min^-1 M^-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (K_p) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K_d value to 571 mM from the native enzyme (K_d = 150 mM). Although arginine-modified enzyme reacts with H₂O₂ to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min^-1 M^-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H₂O₂ suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.     

Thiolation of low-Mr phosphotyrosine protein phosphatase by thiol-disulfides

Thiol-disulfides cause a time- and a concentration-dependent inactivation of the low-M(r) phosphotyrosine protein phosphatase (PTP). We demonstrated that six of eight enzyme cysteines have similar reactivity against 5,5'-dithiobis(nitrobenzoic acid): Their thiolation is accompanied by enzyme inactivation. The inactivation of the enzyme by glutathione disulfide also is accompanied by the thiolation of six cysteine residues. Inorganic phosphate, a competitive enzyme inhibitor, protects the enzyme from inactivation, indicating that the inactivation results from thiolation of the essential active-site cysteine of the enzyme. The inactivation is reversed by dithiothreitol. Although all PTPs have three-dimensional active-site structures very similar to each other and also have identical reaction mechanisms, the thiol group contained in the active site of low-M(r) PTP seems to have lower reactivity than that of other PTPs in the protein thiolation reaction.     

ON THE INACTIVATION OF ASCORBIC ACID OXIDASE

1. In the absence of protective agents, highly purified ascorbic acid oxidase is rapidly inactivated during the enzymatic oxidation of ascorbic acid under optimum experimental conditions. This inactivation, called reaction inactivation to distinguish it from the loss in enzyme activity that frequently occurs in diluted solutions of the oxidase prior to the reaction, is indicated by incomplete oxidation of the ascorbic acid as measured by oxygen uptake; i.e., "inactivation totals." 2. A minor portion of the reaction inactivation appears to be due to environmental factors such as rate of shaking of the manometers, pH of the system, substrate concentration, and oxidase concentration. The presence of inert protein (gelatin) in the system ameliorates the environmental inactivation to a considerable extent, and variation of the above factors in the presence of gelatin has much less effect on the inactivation totals than in the absence of gelatin. 3. A major portion of the reaction inactivation of the oxidase appears to be due to some factor inherent in the ascorbic acid-ascorbic acid oxidase-oxygen system, possibly a highly reactive "redox" form of oxygen other than H₂O₂ or H₂O. The inactivation cannot be attributed to dehydroascorbic acid, the oxidation product of ascorbic acid. 4. Small amounts of native catalase, native peroxidase, native or denatured methemoglobin, and hemin when added to the system, markedly protect the oxidase against inactivation. Cytochrome c has no such protective action. Likewise proteins such as egg albumin, gelatin, denatured catalase, or denatured peroxidase show no such protective action. 5. None of the protective agents mentioned above affect the initial rate of oxygen uptake or change the total oxygen absorbed for complete oxidation of the ascorbic acid, and hence do not act by removal of hydrogen peroxide, per se. 6. Sodium azide and hydroxylamine hydrochloride which inhibit catalase and peroxidase activity also inhibit the protective action of these iron-porphyrin enzymes.     

The reactions of pyridoxal 5′-phosphate with the M4 and H4 isoenzymes of pig lactate dehydrogenase

The H₄ and M₄ isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5′-phosphate. In the early stages, inactivation is largely reversible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5′-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Neither pyruvate nor lactate alone gives any protection. With the M₄ isoenzyme, complete protection against inactivation by pyridoxal 5′-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H₄ isoenzyme. In the course of irreversible inactivation of H₄ lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to resolve earlier controversy.