Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200 microM, with a kmax of 0.111 min-1 and KI = 185 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.     

S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.     

3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.     

Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.     

Role of cysteine and 4′-phosphopantetheine in the inactivation of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme a

S-(4-bromo-2,3-dioxobutyl)-CoA has been used as an inhibitor of fatty acid synthetase from pigeon liver. This affinity label selectively and irreversibly inhibits the acetyl transacylase and β-ketoacyl synthetase reactions of this multienzyme complex. Binding studies with [³H]-labeled bromodioxobutyl-CoA have established that four mol of the inhibitor are bound per mol of the enzyme complex, and that the radioactivity of this compound is covalently bound to cysteine and 4′-phosphopantetheine moieties. Other partial reactions of fatty acid synthesis are unaffected by bromodioxobutyl-CoA.     

Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K(R)) of 99 +/- 2 microM and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17beta-estradiol-3, 17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2.     

Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.     

6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate: new affinity labels for purine nucleotide sites in proteins

Two new adenine nucleotide analogues have been synthesized and characterized: 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate. The bromoketo and dioxobutyl moieties have the ability to react with the nucleophilic side chains of several amino acids, as well as with arginine. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate reacts irreversibly with rabbit muscle pyruvate kinase, causing inactivation. Addition of ADP to the reaction mixture (in the presence of Mg2+) markedly decreases the rate of inactivation. Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP, which reduces the Km for isocitrate. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate reacts irreversibly with isocitrate dehydrogenase, causing, rapidly, a loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and, more slowly, inactivation. Addition of ADP to the reaction mixture (in the presence of Mn2+) protects this enzyme against the loss of allosteric activation. It is proposed that the 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenine nucleotides react at the active site of pyruvate kinase and at the ADP activating site of isocitrate dehydrogenase and that these compounds may have general applicability as affinity labels of catalytic and regulatory adenine nucleotide sites in proteins.     

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeling of pig lung glutathione S-transferase pi by 4-(fluorosulfonyl)benzoic acid

The compound 4-(fluorosulfonyl)benzoic acid (4-FSB) functions as an affinity label of the dimeric pig lung pi class glutathione S-transferase yielding a completely inactive enzyme. Protection against inactivation is provided by glutathione-based ligands, suggesting that the reaction target is near or part of the glutathione binding site. Radioactive 4-FSB is incorporated to the extent of 1 mol per mole of enzyme subunit. Peptide mapping revealed that 4-FSB reacts with two tyrosine residues in the ratio 69% Tyr7 and 31% Tyr106. The ratio is not changed by the addition of ligands. The results suggest that only one of the tyrosine residues can be labeled in the active site of a given subunit; i.e., reactions with Tyr7 and Tyr106 are mutually exclusive. We propose that the difference in labeling of these tyrosine residues is related to their pKa values, with Tyr7 exhibiting the lower pKa. The modified enzyme no longer binds to a S-hexylglutathione-agarose affinity column, even when only one of the active sites contains 4-FSB; these results may reflect interaction between the subunits. We conclude that Tyr7 and Tyr106 of the pig lung class pi glutathione S-transferase are important for function and are located at or close to the substrate binding site of the enzyme.     

On the reaction mechanism of class Pi glutathione S-transferase

Theoretical calculations were performed to examine the ionization of the phenolic group of Tyr7 and the thiol group of glutathione in aqueous solution and in the protein class-pi glutathione S-transferase (GST-Pi). Three model systems were considered for simulations in the protein environment: the free enzyme, the complex between glutathione and the enzyme, and the complex between 1-chloro-2.4-dinitrobenzene, glutathione, and the enzyme. The structures derived from Molecular Dynamics simulations were compared with the crystallographic data available for the complex between the inhibitor S-(p-nitrobenzyl)glutathione and GST-Pi, the glutathione-bound form of GST-Pi, and the free enzyme carboxymethylated in Cys47. Free-energy perturbation techniques were used to determine the thermodynamics quantities for ionization of the phenol and thiol groups. The functional implications of Tyr7 in the activation of the glutathione thiol group are discussed in the light of present results, which in agreement with previous studies suggest that Tyr7 in un-ionized form contributes to the catalytic process of glutathione S-transferase, the thiolate anion being stabilized by hydrogen bond with Tyr7 and by interactions with hydrating water molecules. Proteins 28:530–542, 1997 © 1997 Wiley-Liss, Inc.     

Plasmodium falciparum glutathione S-transferase--structural and mechanistic studies on ligand binding and enzyme inhibition

Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.     

Glutathione S-transferase Pi has at least three distinguishable xenobiotic substrate sites close to its glutathione-binding site

Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (K(I)) of hGST P1-1 on [BITC] is hyperbolic, with K(I) = 66 +/- 7 microM. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr(103) and Cys(47) are modified equally. S-Methylglutathione reduces modification of Cys(47), indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr(103), suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in K(m) for BITC and binds ANS poorly, whereas Y103F has a normal K(m) for BITC and K(d) for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.     

Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase.

The role of the hydroxyl group of tyrosine 6 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione S-transferase has been examined by x-ray crystallography and site-specific replacement of the residue with phenylalanine and evaluation of the catalytic properties of the mutant enzyme. This particuar tyrosine residue is conserved in the sequences of all of the cytosolic enzymes and is found, in crystal structures of both isoenzyme 3-3 from the mu-gene class and an isoenzyme from the pi-gene class, to be proximal to the sulfur of glutathione (GSH) or glutathione sulfonate bound at the active site. The 2.2-A structure of the binary complex of isoenzyme 3-3 and GSH indicates that the hydroxyl group of Tyr6 is located 3.2-3.5 A from the sulfur of GSH, well within hydrogen bonding distance. Removal of the hydroxyl group of Tyr6 has essentially no effect on the dissociation constant (22 +/- 3 microM) for GSH. Nevertheless the Y6F mutant exhibits a turnover number which is only about 1% that of the native enzyme when assayed at pH 6.5 with either 1-chloro-2,4-dinitrobenzene (CDNB) or 4-phenyl-3-buten-2-one. UV difference spectra of the binary enzyme-GSH complexes suggest that the predominant ionization state of GSH in the active site of the Y6F mutant is the neutral thiol (e.g. EY6F.GSH) which is in contrast to the native enzyme in which the thiol is substantially deprotonated (e.g. E.GS-). Spectrophotometric titration suggests that the pKa of the thiol is 6.9 +/- 0.3 in the E.GSH complex and greater than or equal to 8 in the EY6F.GSH binary complex. In addition, the pH dependence of kcat/KmCDNB reveals that the reactions catalyzed by the native enzyme and the Y6F mutant are dependent on a single ionization in the E.GSH and EY6F.GSH complexes with pKa = 6.2 +/- 0.1 and 7.8 +/- 0.3, respectively. The results suggest that the hydrogen bond between Tyr6 and the enzyme-bound nucleophile helps to lower the pKa of GSH in the binary enzyme-substrate complex.     

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.     

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.     

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.     

Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone

Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms.