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A study was conducted to identify the specific cysteine residues involved in the protein S-glutathiolation of complex I at both the 51 kDa and 75 kDa subunits of NQR with a combination of immunoblotting and mass spectrometry. The results implies that S-glutathiolation of the 75 kDa subunit might play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when complex 1 is exposed to redox change in the GSH pool.

Glutathione S-transferase pie (GST pie) is shown to reactivate oxidized 1-cysteine peroxiredoxin (1-cys Prx, Prx VI, Prdx6 and AOP2). A heterodimer complex is formed where 1-Cys Prx is fully active towards either H2O2 or phospholipid hydroperoxide, while the GST pie activity is -25% of that of the GST pie homodimer.

RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents. The effect of a series of deletion mutant proteins on hydrophobicity and transport activity is studied and RLIP76 or one of its derived mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol.

The catalytic efficiency for glutathione (GSH) conjugation reaction with the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is studied structurally for alpha class human GSH transferase (GST) A1-1 [hGSTA1-1], and A2-2 [hGSTA2-2]. Mutational modifications through amino acid substitution in the hGSTA1-1 and hGSTA2-2 structures depicted many fold increase in the catalytic efficiency for (+)-anti-BPDE-GSH conjugation reaction.

A detailed characterization of the N-ethylmaleimide (NEM)-activated microsomal glutathione transferase 1 (MGST1) with respect to the steady state kinetics, the kinetics of thiolate formation and chemical catalysis is presented to fully characterize the mechanistic consequences of activation. Compared to the unactivated enzyme, the activation of MGST1 gives rise to a significantly enhanced efficiency for the utilization of glutathione (GSH).

Research has been conducted on the compound 3-methyleneoxindole (MOI), a plant auxin indole-3-acetic acid's photooxidation product. The experiments indicate that MOI reacts preferentially with glutathione S-transferase pi.

Research has been conducted on the glutathione reductase catalysis. The reduction of the glutathione disulfide by NADPH has been investigated.

The article discusses molecular mechanism of folding of ureadenatured homodimeric human glutathione transferase A1-1. The role of the C-terminus helix 9 as a structural determinant has been discussed.

Ceruloplasmin (CP) is a blue, multicopper alpha2-glycoprotein found in the plasma of all vertebrates. Known for its ability to oxidize ferrous iron and aromatic amines, human CP is also believed to have the ability to catalyze the glutathione-dependent reduction of alkyl hydroperoxide. This peroxidase activity of human CP is shown to be governed by an active site that is distinct from the copper complex site that determines ferroxidase activity.

Two variants of human class pi glutathione (GSH) S-transferase 1-1 with either isoleucine or valine in position 104 (hGSTP1-1(I104) and hGSTP1-1(V104)) have distinct activity toward (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene ((+)-anti-BPDE). The crystal structures of hGSTP1-1(I104)*GSBpd and hGSTP1-1(V104)*GSBpd showed that the half-hydrophilic and half-hydrophobic nature of the H-site is responsible for the enantioselectivity of hGSTP1-1 for (+)-anti-BPDE over (-)-anti-BPDE.

A new photoactivable agonist of nicotinic acetylcholine receptor capable of alkylation of the acetylcholine binding sites was synthesized and developed with the goal of mapping the functional activated site. Results showed that subsequent addition of the oxidant ceric ion Ce(IV) and reduced glutathione before the photolabeling step decreased substantially nonspecific labeling without affect the binding characteristics of the acetylcholine binding sites.

A mechanism for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B) is the glutathionylation of the active site cysteine 215. Mass spectrometry reveals that treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide causes modification in which the cysteine residues are oxidixed to mixed disulfides with glutathione. Results also indicate that the inactivated form of the phosphatase is a glutathionyl mixed disulfide.

An experiment was undertaken to investigate the pH dependence of the oxidation-reduction potential for Escherichia coli and human glutathione reductase using high-resolution X-ray structures and compared the redox potential with the yeast enzyme without a high resolution structure. The redox potentials used in the study were the oxidized and the two-electron reduced enzyme. It was revealed that glutathione reductase declined its activity upon deprotonation of the acid-base catalyst at a high pH and the pKa values were associated with the two-electron reduced enzyme transition.

The thiol esterase activity of the gluthathione S-transferase A1-1 (GSTA1-1) on the thiol ester produced from glutathione (GSH) and ethacrynic acid (EA) was examined. GSTs play a vital role in detoxification and in GSH-dependent tumor cell resistance to electrophilic chemotherapeutic agents. It was observed that the GSTA1-1 isoform from rat activated the hydolysis of the non-carbamate thiol ester glutathionyl ethacrynate and that GST-catalyzed hydrolysis of thiol esters led to the distribution and steady state levels of free acid and GSH conjugate in remote sites of synthesis.

A study was conducted to describe the equilibrium and kinetic unfolding features of homodimeric class alpha glutathione transferase A1-1 (hGST A1-1). The enzyme activity of native and alkylated hGST A1-1 was spectrophotometrically evaluated at 340 min in a 0.1 M potassium phosphate buffer solution. Results indicated that the equilibrium unfolding of hGST A1-1 correlated with the two-state transition between native dimer and unfolded monomer.

A study was conducted to examine the reconstitution of purified dinitrophenyl S-glutathione (DNP-SG) ATPase into artificial liposomes. ATP-dependent DNP-SG transport kinetics were analyzed at DNP-SG concentrations from 14 to 170 mu-M while initial velocity measurements for doxorubicin transport were determined using 14-(14C)DOX concentrations from 2 to 6 mu-M. ATP Results indicated that doxorubicin is a competitive inhibitor of DNP-SG transport.

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an 'atypical' crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.

The fate of the thiol proton coming from the ionization of the sulfhydryl group of GSH in the active site of glutathione transferase P1-1 has been studied. pH changes caused by the binding of GSH to the enzyme in the absence of any inorganic buffer indicate that the thiol proton leaves the active site when the binary complex is formed. The amount of protons released is stoichiometric to the amount of GSH thiolate formed in the G-site. The apparent [pK.sub.a] value for the bound GSH, calculated with this potentiometric approach, is 6.18 [+ or -] 0.09; very similar values are found by spectrophotometric (6.20 [+ or -] 0.12) and by kinetic (6.00 [+ or -] 0.08) experiments. Binding of S-hexylglutathione does not cause any proton release. Stopped-flow data obtained by means of an acid-base indicator show that the proton extrusion process (apparent [t.sub.1/2] = 1.1 [+ or -] 0.1 ms at 15 [degrees] C) is not rate limiting in turnover (apparent [t.sub.1/2] = 34 [+ or -] 4 ms at 15 [degrees] C). By comparing the kinetic behavior of three distinct events occurring during the binding of GSH to the enzyme, i.e., proton release, ionization of bound GSH and quenching of intrinsic fluorescence, it appears that the binding process follows a multistep mechanism possibly involving the conformational transition of a weak precomplex into the final Michaelis complex. This step is modulated by helix 2 motions and may be rate limiting at physiological GSH concentrations. These findings, coming from kinetic studies, are consistent with NMR data [Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A.M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027] and time-resolved fluorescence experiments [Stella, L., Caccuri, A.M., Rosato, N., Nicotra, M., Lo Bello, M., De Matteis, F., Mazzetti, A. P., Federici, G., and Ricci, G., manuscript in preparation].

Kinetic and structural alterations in recombinant human aldose reductase (AR) because of modification by S-nitrosoglutathione (GSNO) were investigated. Findings showed that such modification inactivates AR. The nitrosothiol GSNO was formed by aerobic modification of GSH by NO which has been proposed as a mediator of some of the biological effects of NO. Because high concentrations of GSH are found in most cells, GSNO most likely supports the NO transfer reactions to sulfhydryl centers of cytosolic proteins.

The double mutant of human glutathione reductase (GRTR) and its glutathione, trypanothione and glutanoyl spermidine complexes were analyzed to determine their structure and engineered change in substrate specificity. Results reveal that the mutation of glutathione reductase into trypanothione reductase causes steric hindrance, which leads to a large displacement of the side chain of Arg 347. Furthermore, this displacement and the change in charge introduced by the mutation modify the binding cavity that forces glutathione to adopt a nonproductive mode and glutathionylspermidine to assume a productive mode.

The crystal structure generated by the complexation of Escherichia coli B glutathione synthetase (GSHase) to ADP, glutathione, two Mg(sup 2+) and one sulfate ion bound at the active site was obtained at 2 angstrom. The structure, which is a tetramer with 316 amino acid residues in each subunit, represents a pseudo-Michaelis complex in the reverse reaction of GSHase due to the chemical similarities between phosphate and sulfate.

The solid-state kinetics, site-directed mutagenesis, fluorescence and ultraviolet spectroscopies, ab initio calculations and molecular modeling were employed to examine the cofactor glutathione (GSH) in contact with the active site of rat A1-1 glutathione S-transferase. Specifically, it was found that the pKa of the cofactor decreases after PHe-220 was replaced by Tyr-9 due to the generation of an on-face Ar-OH-pi hydrogen bond.

The participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1 is suggested. Studies reveal that the alpha-carboxylate of the Glu residue of glutathione serves as a proton acceptor in the catalytic process. The glutathione molecule not only acts as a substrate for the thiol group but also as a substrate contributing a carboxylate that serves as a proton acceptor in the catalytic process and a carboxylate which modulates binding of the second substrate to the enzyme.

Experimental results on the kinetics of disulfide-bonded ribonuclease (RNase) T1 synthesis shows that high yields of RNase T1 can be obtained under various redox conditions. The mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) was used as a homogenous starting material, and folding was induced by adding free glutathione. Addition of protein disufide isomerase catalyzes the refolding process and enhances the yield and rate of reaction. The hsp homolog BiP inhibits the folding of GS-RNase T1 in the absence of ATP, and addition of Mg-ATP restores refolding.

Fluorescence studies of mixed disulfides between glutathione and reduced disulfide-bound proteins revealed that the GS-S-protein-combined disulfide derivatives act as unfolded protein models. These proteins act as substrates, thus validating secretory protein folding and the reaction between unfolded proteins and protein-folding catalysts. GS-RNase T and GS-alpha-lactalbumin induce ATPase activity in BiP.

Glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl) contain a complex known as glutathionylcobalamin (GSCbl). The cobalamin coenzymes use this as a pathway in their formation. The total 1H and 13C NMR spectra of GSCbl have been examined by various methods, including homonuclear and inverse detected heteronuclear two-dimensional NMR methods. The cystein-alpha and -beta positions reveal the largest variation when the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl are compared to the shifts of free GSH and GS-, showing that the cysteine sulfur atom is used to conduct GSH to the cobalt atom in GSCbl.

The redox system found in all organisms includes the flavoenzyme thioredoxin reductase (TR) and its natural thioredoxin substrate. TR is purified from the human placenta as a dimer of 60 kDa subunits for the determination of the biochemical properties of the redox system. Gel filtration chromatography estimate the molecular size of native TR as 160 kDa, while the migration on a sucrose gradient has a molecular mass of 130 kDa.

Wild-type human glutathione reductase (hGR) and inactive mutants with single or double mutations at residues 446 and 447 were isolated and characterized. The mutant G446E/F447P-hGR behaved like a poorly folded monomeric protein. The results suggest that the sequence around G446 can control dimerization as well as domain folding. The role of interface residues as a folding signal and the prospect to develop dimerization inhibitors of hGR as antiparasitic drugs are discussed.

Multiple isomorphous replacement methods were used to determine the crystal structure of a glutathione S-transferase from the mu class complexed with glutathione at a resolution of 2.2 angstroms (A). The enzyme underwent crystallization in the monoclinic space group C2 and acquired unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A and beta = 106.07degrees. A final model of the protein, obtained by interpreting part of the solvent structure, reveals that it was made up of 4150 atoms, which included the non-hydrogen atoms belonging to 434 amino acid residues, two glutathione molecules, and oxygen atoms of 474 water molecules.

Treatment of nondisplaceable radioligand binding with a glutathione (GSH) metabolism inhibitor, borate with L-serine, has allowed kinetic characterization of /(super 35)S~GSH binding to hydra membranes. The rapid, reversible and saturable GSH binding to intact membranes has a dissociation constant comparable to that of GSH binding to detergent-solubilized membranes. Thus, the solubilized preparation holds promise for further characterization and purification of the putative glutathione chemoreceptor.

Glutathione is involved in the mobilization and delivery of Cu(I) for its biosynthesis. The Cu(I)-GSH complex acts in vitro as the Cu(I) source in the reconstitution of American lobster, Homarus americanus, apohemocyanin. The binding of Cu(I) to the high-affinity external binding site of the hemocyanin oligomer facilitates the incorporation of copper into the protein matrix. Conversion into biologically active, binuclear copper sites occurs immediately after the formation of mononuclear copper sites.

The domain and catalytic core of Tn501 MerA as separate proteins is cloned and expressed to simplify functional analysis of NmerA. Tn501 NmerA is demonstrated to be a stable, soluble protein that binds 1 Hg(super 2+)/domain and delivers it to the catalytic core at kinetically competent rates.

The thiol exchange reaction is investigated using rapid flow-quench kinetic studies of the reaction between thiols and ESSG, the form of the enzyme in which glutathione is covalently attached to Cys13. The results suggest that the second glutathione binds very weakly and that its pK(super a) is not significantly perturbed from that observed in solution.

The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octyglutathione (S-octyl-GSH), when combined with fluorescence spectroscopy and thermodynamic data, allow for detailed characterization of the ligand-binding process.

Site-directed mutagenesis is employed in alpha class glutathione S-transferase, isozyme A1-1, to investigate the hydrophobic ball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit by Met94, Phe136 and Val139, and the Arg/Glu region consisting of Arg69 and Glu97 from both subunits. The aim of the study is to evaluate the importance of each at the subunit interface and to determine the generation of monomeric enzymes by such single mutations.

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP) was synthesized and designed as a new photoaffinity label for glutathione S-transferase. The substrate analogue retained glutathione moiety which is expected to direct it to the active site of glutathione S-transferase. It featured the photoreactive benzophenone group which can react with carbon-hydrogen bonds of either side chain or enzyme backbone. Results demonstrated that modification of a methionyl residue on one subunit prevents reaction at a second methionine on the other subunit of the analogue.

Research was conducted to determine the X-ray crystal structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione (HIPC-GSH) and with NBC-GSH. Based on the comparison of the three structures, it is apparent that whereas His 126 and Glu 33 interact tightly with the metal ion in a fixed conformation, the other ligands are reasonably flexible and can move to accommodate the various inhibitors. Results are consistent with an ionic rather than a covalent nature of the interactions between the carboxylates and the zinc ions.

Research was conducted to investigate the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site. The enzyme structure model was derived using homology modeling. The redesign of the protein was based on the unique structural characteristics of the enzyme to examine their importance for substrate binding and catalysis and to find if an enzyme with a higher catalytic efficiency could be achieved.

The cysteine protease known as prohormone thiol protease (PTP) was determined to be a key proenkephalin processing enzyme in secretory vesicles of adrenal medulla. It is shown that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione found in chromaffin granules. Research findings also showed that the electrophoretic mobilities of the four subunits differed upon reducing compared to nonreducing conditions.

A study was conducted to analyze the molecular mechanism by which the C-terminus affects substrate binding, product release or an intermediate step of the conjugation reaction of glutathione S-transferase. Steady-state and a pre-steady-state kinetic analyses were carried out. Results indicated that the ligand exists as a diastereomeric mixture. Findings also showed the presence of a single relaxation rate.

The histidine residue at position 107 (His107) of the human subunit histidine glutathione S-transferases (GST) has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions as revealed in domain interchange analyses and site-directed mutagenesis. His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation.

The molecular aspects of human thioltransferase specificity for the glutathione moiety were investigated. A quadruple Cys to Ser mutant of human thioltransferase, namely, C7S, C25S, C78S and C82S, was derived. Experimental results showed that the disulfide-adducted glutathione in the thioltransferase-S-S-glutathione complex has an extended conformation and is localized in a cleft near the protein surface containing the residues from helices-alpha2, alpha3, the active site loop and the loop linking helix-alpha3 and strand-beta3.