Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involvement of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K(obs) of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase.     

Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.     

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.     

The effect of radiofrequency energy on the length and temperature properties of the glenohumeral joint capsule

Two isozymes of mammalian methionine adenosyltransferase, MAT I and MAT III, are expressed solely in adult liver. They are, respectively, tetramers and dimers of a single subunit encoded by the gene MAT1A. A third isozyme, MAT II, contains a catalytic subunit encoded by a separate gene, MAT2A, and is expressed in a variety of tissues, including (to a slight extent) adult liver. Based on a recent finding that 2 children with isolated hypermethioninemia and brain demyelination were homozygous for MAT1A mutations predicted to produce severely truncated proteins, and devoid of activity when expressed, it was concluded that complete lack of MAT I/III activity may be associated with neurological symptoms and demyelination. We now report that a 43-year-old man with persistent isolated hypermethioninemia, previously demonstrated to have deficient MAT activity in his liver, has normal brain myelination on MRI and normal neurological function, despite being homozygous for a 539 TG insertion in exon V of MAT1A, so that the gene is predicted to encode a protein of only 184 rather than the normal 395 amino acids. This patient's exon V mutation was demonstrated by SSCP analysis and verified by sequencing. Both parents are heterozygous for the same insertion. This suggests that MAT1A mutations producing severely truncated proteins do not necessarily produce brain demyelination. This finding has relevance to a previously reported 4-year-old girl who was also homozygous for the 539insTG mutation. Finally, our patient's 7% residual hepatic MAT activity, measured at 1 mM methionine, may reflect the hepatic activity of the more ubiquitous enzyme form, MAT II.     

Generation of free radicals from hydrogen peroxide and lipid hydroperoxides in the presence of Cr(III)

Free radical generation from H2O2 and lipid hydroperoxides in the presence of Cr(III) was investigated by electron spin resonance (ESR) spin trapping methodology. Incubation of Cr(III) with H2O2 at physiological pH generated hydroxyl (.OH) radical, the yield of which reached saturation level in about 6 min. Deferoxamine reduced the .OH radical yield by only about 20%, diethylenetriamine pentaacetic acid (DTPA) reduced it by about 70%, while cysteine, glutathione, and NADH exhibited no significant effect. The yield of .OH radical formation also depended on the pH being 15 times higher at pH 10 than that at pH 7.2. At pH 3.0, .OH radical generation became nondetectable, and addition of H2O2 to Cr(III) solution did not affect the intensity of the Cr(III) ESR signal while at pH 10, addition of H2O2 reduced the Cr(III) intensity by about 40%, showing that reaction of Cr(III) with H2O2 occurred only at higher pH. Incubation of Cr(III) with the model lipid hydroperoxides, cumene hydroperoxide and t-butyl hydroperoxide, generated lipid hydroperoxide-derived free radicals. Addition of deferoxamine or DTPA had a minor inhibitory effect on that generation. These results show that Cr(III) is capable of producing free radicals from H2O2 and lipid hydroperoxides, which may have significant implications regarding the mechanism of chromium-induced carcinogenesis.     

S-adenosylmethionine synthesis: molecular mechanisms and clinical implications

Methionine adenosyltransferase (MAT) is an ubiquitous enzyme that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. In mammals, there are two genes coding for MAT, one expressed exclusively in the liver and a second enzyme present in all tissues. Molecular studies indicate that liver MAT exists in two forms: as a homodimer and as a homotetramer of the same oligomeric subunit. The liver-specific isoenzymes are inhibited in human liver cirrhosis, and this is the cause of the abnormal metabolism of methionine in these subjects.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.     

Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.     

Differential effects of diazepam on anxiety in streptozotocin induced diabetic and non-diabetic rats

In the absence of added Fe2+, the ATPase activity of isolated Schizosaccharomyces pombe plasma membranes (5-7 mumol P(i) per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner. Sizable inactivation occurs only at 50-80 mmol/L H2O2. The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both the K(m) of the enzyme for ATP and the V of ATP splitting. On exposing the membranes to the Fenton reagent (50 mumol/L Fe2+ + 20 mmol/L H2O2), which causes a fast production of HO. radicals, the ATPase is 50-60% inactivated and 90% of added Fe2+ is oxidized to Fe3+ within 1 min. The inactivation occurs only when Fe2+ is added before H2O2 and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO. radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO. production.     

Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site

We describe a type of mild hypermethioninemia due to a point mutation in the MATA1 gene, which was inherited dominantly in a family. Three patients coming from the same family pedigree were detected by the presence of isolated hypermethioninemia on a mass-screening program. The measurement of methionine adenosyltransferase (MAT) activity in a patient's liver revealed a partial deficiency of hepatic MAT with a reduction in the Km for methionine. Single strand conformation polymorphism (SSCP) analysis and direct sequencing of the patients' genomic DNA revealed a G to A mutation at nucleotide 791 that converts Arg-264 to His (R264H) in one allele of MATA1 gene. The other allele was normal in all the patients examined. Gene tracking in the family revealed that the hypermethioninemia is associated with heterozygosity for the R264H mutation in the MATA1 gene.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo

Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.     

Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.     

Lipoxygenase-catalyzed oxidation of chlorpromazine by hydrogen peroxide at acidic pH

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidizes chlorpromazine using H2O2 at acidic pHs ranging from 3.0 to 4.0. The enzyme is assayed at pH 3.5, at which the half-life is 2 h (lower pHs cause higher inactivation rates). This oxidation is enzymatical since boiled enzyme or even iron ions both with H2O2 failed to produce any increase in absorbance. In addition, the concentration of CPZ radical cation formed and the concomitant enzyme activity directly depends on the enzyme concentration up to 0.23 microM. The Vmax value is 125 mumol/min per mg protein and the Km for chlorpromazine and H2O2 are 2.1 mM and 0.25 mM, respectively. Similar results were obtained when linoleic acid hydroperoxide was used instead of H2O2 with a Km value of 95 microM. The radical cation obtained in the oxidation of chlorpromazine by lipoxygenase decays by a disproportionation reaction. This permits to consider the overall reaction as a sum of an enzymatic reaction coupled with a chemical second order reaction with substrate regeneration, similar to those produced by peroxidases from different sources.     

Thiols as mechanistic probes for catalysis by the free radical enzyme galactose oxidase

Galactose oxidase, a mononuclear copper enzyme, oxidizes primary alcohols to aldehydes using molecular oxygen. A unique type of cross-link between tyrosine 272, an active-site copper ligand, and cysteine 228 provides a modified tyrosine radical site believed to act as a one-electron redox center. Substrate analogs incorporating a primary thiol group in place of the primary alcohol group in normal substrates (RCH2OH) have been studied as active-site mechanistic probes. Thiol sulfur coordinates to the active-site copper, leading to enzyme inactivation in a time- and concentration-dependent manner. The mechanism of inactivation involves redox chemistry related to the active-site redox centers, though inactivation does not proceed through the rate-determining hydrogen atom abstraction step that occurs in alcohol oxidation. Thiols are therefore classified as active-site-directed redox inactivators. The thiol analog of galactose, 6-Thio-Me-Gal, is also turned over by the enzyme, albeit at a much reduced rate, indicating that the energetics of turnover is changed significantly. Thiols constitute a particularly good model of the ground state enzyme-substrate complex. The Michaelis complex for thiol substrate analogs is stabilized at least 200-fold compared to the analogous alcohol substrates, whereas the transition state of H atom abstraction is destabilized, presumably due to a slight increase in distances of reacting atoms and weakening of hydrogen-bonding interactions due to the larger atomic radius of sulfur compared to that of oxygen.     

Dopamine inactivates tryptophan hydroxylase and forms a redox-cycling quinoprotein: possible endogenous toxin to serotonin neurons

Exposure of tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, to dopamine under mild oxidizing conditions (iron + H2O2) or in the presence of tyrosinase results in a concentration-dependent inactivation of the enzyme. Dopamine, iron, H2O2, or tyrosinase alone does not alter TPH activity. Similarly, N-acetyldopamine oxidized with one equivalent of sodium periodate causes a concentration-dependent inactivation of TPH as well. TPH is protected from dopamine-induced inactivation by reduced glutathione, ascorbic acid, and dithiothreitol but not by the radical scavengers DMSO, mannitol, or superoxide dismutase. Parallel studies with [3H]dopamine reveal a high negative correlation between inhibition of catalysis and incorporation of tritium into the enzyme. Those reducing agents and antioxidants that protect TPH from inactivation are effective in preventing the labeling of TPH by [3H]dopamine. Acid hydrolysis and HPLC with electrochemical detection (HPLC-EC) analysis of inactivated TPH revealed the formation of cysteinyl-dopamine residues within the enzyme. Exposure of dopamine-modified TPH to redox-cycling staining after SDS-PAGE confirmed the formation of a quinoprotein. These results indicate that dopamine-quinones covalently modify cysteinyl residues in TPH, leading directly to the loss of catalytic activity, and establish that TPH could be a target for dopamine-quinones in vivo after drugs (e.g., neurotoxic amphetamines) that cause dopamine-dependent inactivation of TPH. Redox cycling of a TPH-quinoprotein could also participate in the serotonin neuronal toxicity caused by these same drugs.     

Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.     

Reactions of bovine liver catalase with superoxide radicals and hydrogen peroxide

The oxidized intermediates generated upon exposure of bovine liver catalase to hydrogen peroxide (H2O2) and superoxide radical (O2-) fluxes were examined with UV-visible spectrophotometry. H2O2 and O2- were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible (450-700 nm) regions showed that three oxidized intermediates, namely Compounds I, II and III, can be observed upon exposure of catalase to enzymatically generated H2O2 and O2-. Compound I is formed during the reaction of native enzyme with H2O2 and disappears in two ways: (i) via the catalytic reaction with H2O2 to restore native catalase and (ii) via the reaction with O2- to form Compound II. At low H2O2 concentrations (< 4.8 x 10(-9) M H2O2), Compound II reverts towards the native state mainly in a direct one-step reaction, whereas at higher H2O2 concentrations the pathway of Compound II back to the native enzyme involves Compound III. Formation of the latter from Compound II and H2O2 is irreversible and the rate constant of this reaction is 6.1 +/- 0.2 x 10(4) M-1 s-1. The formation of Compound III through the direct reaction of O2- with native enzyme has also been observed. Depending on the experimental conditions, the inactivation of catalase by O2- can be due to accumulation of Compound II ("slow" inhibition) or to the formation of Compound III ("rapid" inhibition) part of which leads to a dead end product. Formation of Compound III and of this dead end product are responsible for the irreversible inactivation in presence of an excess of H2O2.     

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.