Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Application of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate for detection of cystine-containing peptides

A simple, sensitive and reliable method for the detection of cystine-containing peptides has been developed. A peptide bridged with a disulfide bond was reduced and cleaved with tributylphosphine, and then coupled with a thiol specific reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, under alkaline conditions. After incubation at 60 degrees C for 1 h, the fluorescent derivative formed was measured with excitation at 385 nm and emission at 515 nm. The intensity of fluorescence labeled to the peptide was very stable and the peptide containing disulfide was quantitatively determined in the range of 100 pmol to 10 nmol, when oxidized glutathione was used as a standard. This method was very useful for specific detection of cystine-containing peptides in the column effluents on reverse phase high performance liquid chromatography.     

Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Fluorescence properties of o-phthaldialdehyde derivatives of amino acids.

The fluorescence properties of the products formed by reaction of o-phthaldialdehyde with amino acids and their derivatives, in the presence of thiol compounds, have been studied. The emission spectra, quantum yields, and lifetimes depend on the primary amine and thiol compound used; the observations confirm the report (Simsons, S.S., Jr. and Johnson, D.F. (1978) J. Org. Chem 43, 2886--2891) that the product incorporates molecules of all three types of compounds. The fluorescence quantum yields of o-phthaldialdehyde derivatives of the naturally occuring amino acids ranged from 0.33 to 0.47, using 2-mercaptoethanol as the thiol compound. The fluorescence lifetimes were about 18--20 ns. Lower quantum yields were obtained when mercaptoethanol was replaced by dithiothreitol or ethnethiol. Derivatives of amino acid amides and peptides had quantum yeilds as low as 0.03, due to quenching by the carboxamide group. The intramolecular quenching was relieved by the detergent, sodium dodecyl sulfate, and by dimethylsulfoxide. Monosubstituted lysine exhibited a normal fluorescence, but the di-substituted product was largely quenched, presumably due to interaction between the two isoindole fluorophors. Fluorscence stopped-flow experiments showed that the alpha- and epsilon-amino groups reacted at different rates, with the epsilon-amion group reacting 10 times faster, with a t 1/2 of about 6 s under pseudo first order conditions at pH 9.0 with 10(-3) M o-phthaldialdehyde. The amount of instability shown by the o-phthaldialdehyde derivatives depended on the thiol compound used, the primary amine involved, and the solvent. Cysteine and o-phthaldialdehyde reacted to give an unstable, weakly fluorescent product; but cysteine could be assayed normally if its sulfhydryl was blocked. The o-phthaldialdehyde reagent was discussed in relation to fluorescamine, another reagent for primary amines.     

A Multipurpose receptor composed of promiscuous proteins. Analyte detection through pattern recognition

A multipurpose receptor akin to the "electronic nose" was composed of coumarin-labeled mutants of human glutathione transferase A1. We have previously constructed a kit for site-specific modification of a lysine residue (A216K) using a thiol ester of glutathione (GSC-Cou bio) as a modifying reagent. In the present investigation, we scrambled the hydrophobic binding site (H-site) of the protein scaffold through mutations at position M208 via random mutagenesis and isolated a representative library of 11 A216K/M208X mutants. All of the double mutants could be site-specifically labeled to form the K216 Cou conjugates. The labeled proteins responded to the addition of different analytes with signature changes in their fluorescence spectra resulting in a matrix of 96 data points per analyte. Ligands as diverse as n-valeric acid, fumaric acid monoethyl ester, lithocholic acid, 1-chloro-2,4-dinitrobenzene (CDNB), glutathione (GSH), S-methyl-GSH, S-hexyl-GSH, and GS-DNB all gave rise to signals that potentially can be interpreted through pattern recognition. The measured K d values range from low micromolar to low millimolar. The cysteine residue C112 was used to anchor the coumarin-labeled protein to a PEG-based hydrogel chip in order to develop surface-based biosensing systems. We have thus initiated the development of a multipurpose, artificial receptor composed of an array of promiscuous proteins where detection of the analyte occurs through pattern recognition of fluorescence signals. In this system, many relatively poor binders each contribute to detailed readout in a truly egalitarian fashion.     

The effects of temperature and pH on the reaction between 1-chloro-2,4-dinitrobenzene and glutathione, catalyzed by the major glutathione S-transferase from Galleria mellonella

The nucleophilic substitution reaction between glutathione and 1-chloro-2,4-dinitrobenzene has been studied at temperatures between 4 and 42°C and pH values between 6.99 and 10.80. The apparent enthalpy, entropy and free energy of ionization of the thiol group have been estimated as have the apparent enthalpy, entropy and free energy of activation of the reaction between the glutathione thiolate anion and the aromatic electrophile. The results obtained permit the calculation of values of the second order rate constant governing the reaction at a range of temperatures and pHs. These values are in accord with those reported in the literature from experimental work by others. The major glutathione S-transferase from Galleria mellonella has been studied with respect to its kinetic responses to changes in pH and temperature. There appear to be two kinetically critical ionizations governing the reaction at high pH. These ionization events are characterized by apparent pK_a values of 8.61 ± 0.15 and 9.16 ± 0.22. A thermodynamic model of the kinetic behavior of the enzyme permits the prediction of its activity over a range of pH and temperature values. The apparent free energy of activation for the enzyme catalyzed reaction is only 7% lower than that for the non-catalyzed reaction between 1-chloro-2,4-dinitrobenzene and glutathione thiolate anion. This observation is compatible with the suggestion that promotion of the ionization of the glutathione thiol group is the major mechanism of catalysis.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Measurement of distance between the active serine of the thioesterase domain and the pantetheine thiol of fatty acid synthase by fluorescence resonance energy transfer

Fatty acid synthase from the uropygial gland was inactivated by treatment with pyrenebutyl methanephosphonofluoridate by specific modification of the "active serine" at the thioesterase domain. Treatment of fatty acid synthase with 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin resulted in the loss of the condensation activity and overall synthase activity. Acetyl-CoA and malenyl-CoA protected the enzyme from inactivation by this reagent suggesting that the pantetheine thiol was modified. In support of this conclusion was the finding that modification of the primer-binding thiol with iodoacetamide prior to the modification with the coumarin derivative resulted in no change in the binding of the coumarin to the enzyme. Furthermore, the presumptive active site peptide isolated after proteolysis released its attached coumarin upon treatment with alkali under beta-elimination reaction conditions. Graphical analysis of the binding data suggested that binding of one coumarin derivative/subunit of the synthase would result in complete loss of the synthase activity. When the synthase was modified with the coumarin and pyrene derivatives, fluorescence resonance energy transfer occurred from the pyrene at the thioesterase site to the coumarin attached to the pantetheine thiol. Dissociation of the enzyme to monomers did not decrease the efficiency of transfer, but limited trypsin treatment, which released the thioesterase domain, abolished the fluorescence resonance energy transfer. These results suggested that the energy transfer occurred between intrasubunit sites. The distance between the pyrene at the thioesterase active site and the coumarin attached to pantetheine thiol on the same subunit of fatty acid synthase was estimated from the efficiency of energy transfer to be 37 A.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 7: Assay and characterization of 7,8-diacetoxy-4-methylcoumarin:protein transacetylase from rat liver microsomes based on the irreversible inhibition of cytosolic glutathione S-transferase

The enzymatic transfer of acetyl groups from acetylated xenobiotics to specific proteins is a relatively grey area in the evergreen field of biotransformation of foreign compounds. In this paper, we have documented evidence for the existence of a transacetylase in liver microsomes that catalyses the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to glutathione S-transferase (GST), either purified or present in cytosol leading to the irreversible inhibition of GST. A simple procedure is described for the assay of transacetylase by preincubation of DAMC with liver microsomes and pure GST/liver cytosol, followed by the addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) in order to quantify GST activity by the conventional procedure. The extent of inhibition of GST by DAMC under the conditions of the assay is indicative of DAMC:protein transacetylase activity. Following the assay procedure described here, the transacetylase was shown to exhibit hyperbolic kinetics. The bimolecular nature of the transacetylase reaction was apparent by the demonstration of Km and vmax values. 7,8-Dihydroxy-4-methylcoumarin (DHMC), one of the products of transacetylase reaction was identified and quantified using the partially purified enzyme. The fact that p-hydroxymercuribenzoate (PHMB) and iodoacetamide abolished irreversible inhibition of GST upon the action of transacetylase on DAMC strongly characterized transacetylase as a protein containing thiol group at the active site. In addition, the relative specificities of acetoxy 4-methylcoumarins to transacetylase have been demonstrated.     

Mechanism of Antifungal Action of 2,4,5,6-Tetrachloroisophthalonitrile

Studies of the toxicity of 2,4,5,6-tetrachloroisophthalonitrile (TCIN) to cells of Saccharomyces pastorianus and Neurospora crassa showed that 2 to 4 μg/ml of the toxicant were required to inhibit growth. Several thiol compounds reversed toxicity to growth. Glucose oxidation was severely impaired in treated cells of both test organisms. The toxicant at 2 or 4 μg/ml markedly reduced soluble thiol content of these cells. Bound thiol content was less affected in S. pastorianus cells than soluble thiol content. Uptake of toxicant by yeast cells was accompanied by formation of derivatives, some of which resembled those formed by reaction of glutathione with TCIN in vitro. Coenzyme A, glutathione, and 2-mercaptoethanol readily formed derivatives with TCIN in vitro. The nature of these derivatives was studied using 2-mercaptoethanol products as model substituents. Four derivatives were formed with 2-mercaptoethanol each with similar functional groups but showing dissimilar degrees of mobility during silica gel chromatography. Evidence indicated that these are derivatives of TCIN in which 1 to 4 of the halogens has been substituted by 2-mercaptoethanol. The mechanism of fungicidal action of TCIN is attributed to thiol inactivation.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.     

Synthesis of N-(9-Acridinyl)maleimide,a Fluorometrical Reagent for Thiol Compounds

The authors have reported¹⁾ a new maleimide type fluorescent thiol reagent, N-(9-acridinyl)- maleimide (NAM). In this paper the syntheses of NAM and its coupling products with thiol compounds are presented. NAM was synthesized from 9-aminoacridine and maleic anhydride through dehydratic cyclization in polyphosphoric acid. NAM showed no substantial fluorescence but its coupling products with thiol compounds exhibited strong blue fluorescence. Application of NAM for the fluorometrical analysis of cysteine and glutathione are suggested.     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

Study of factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)–thiol derivatives by liquid chromatography

A detailed investigation of the factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)–thiol derivatives (i.e. cysteine, homocysteine and cysteinylglycine) is described. Essentially, this assay entails extracting specific thiols by plasma disulphide bond reduction, protein precipitation, sulphydryl compound derivatization with the thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F), and subsequent separation with isocratic reversed-phase high-performance liquid chromatography. By improving the reliability of several analytical parameters (composition of the mobile phase, pretreatment of the sample using different reducing and protein precipitation agents, and optimization of the derivatization of thiols with SBD-F), a number of critical issues can be identified and solved.     

Modification of acetylcholinesterase with the fluorescent thiol reagent S-mercuric-N-dansylcysteine

11s acetylcholinesterase (EC 3.1.1.7) from Torpedo californica electroplax, purified by a combination of affinity and gel chromatography was found to react stoichiometrically with S-mercuric-N-dansylcysteine. Approximately four mols of reagent per mol of enzyme were incorporated when the modification was carried out in 1.0 mM Tris-C1, pH 7.5, either in the presence or absence of 0.1 M NaCl. Prior incubation of the enzyme with 1.0 x 10(-4) M Zn2+ allowed the incorporation of about six mols of reagent per mol of enzyme. Binding of the reagent produced shifts in the emission and excitation wavelength maxima which were similar for all reaction conditions; however the enhancement of fluorescence intensity which accompanied binding of reagent was dependent on the ionic composition of the reaction medium. The modified enzyme remained active towards the active site titrant 7-(dimethylcarbamoyloxy)-N-methylquinolinium and retained its sensitivity towards inactivation by Zn2+. The results suggest that acetylcholinesterase as prepared contains several accessible thiol groups, and that the bound reagent may prove to be a useful probe of ligand-induced conformational changes in acetylcholinesterase.     

Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase.

The environments of the two sulfhydryl groups of procine muscle adenylate kinase have been investigated by chemical modification reactions. The results indicate that the environments of the two-SH groups of procine muscle adenylate kinase are markedly different and that substrates induce conformational changes in the enzyme in the region of the sulfhydryl groups. The fluorogenic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-chloride) reacts specifically with the -SH groups of the enzyme at pH 7.9. One thiol group reacts with NBD-chloride approximately 40-fold faster than the other one, and the fast reacting group has been identified as Cys-25 in the amino acid sequence. The similarity of the rate of the more slowly reacting Cys-187 with NBD-chloride to that of glutathione with the same reagent is consistent with its location on the surface of the enzyme as determined by x-ray crystallography structure. The fast reacting Cys-25 in the interior of the structure can be approached by compounds such as NBD-chloride via a cleft. Reaction of Cys-25, presumably located close to the catalytic center, leads to complete inactivation of the enzyme. Substrates such as ATP, MgATP, and ADP which bind to the triphosphate subsite of the enzyme decrease the rate of reaction of Cys-25 by factors up to 3.5 but have only a small effect (approximately equal to 10%) on the reactivity of Cys-187. AMP, however, has a pronounced effect on the reactivity of Cys-187, the slowly reacting group. The multisubstrate analogue P-1, P-5-di-(adenosine-5)pentaphosphate (Ap-5A) decreases the rate of reaction of the fast reacting thiol group by a factor of 300. The behavior of Cys-25 toward NBD-chloride, i.e. super-reactivity in the absense of Ap-5A and slow reactivity in the presence of the multisubstrate inhibitor, was characteristic for both porcin and carp adenylate kinase. In the presence of Ap-5A adenylate kinase can be selectively modified at Cys-187; the introduction of the fluorescent NBD group at this position has no effect on enzymatic activity. A slow transfer of the NBD group occurs from the third groups to the epsilon-amino group of Lys-31. This transfer reaction is further evidence that the structure of adenylate kinase in dilute solution is similar to that of the crystalline enzyme since the x-ray data have shown that the sulfur of Cys-187 and the epsilon-nitrogen of Lys-31 are less than 4 A apart. The strongly fluorescent NBD-NH-enzyme possesses full activity and binds substrates as. cont'd     

Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism.

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513--522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.