Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V(max) values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS--PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.     

Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a-1a

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a-1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme were replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys-deficient mutant forms of glutathione transferase M1a-1a were catalytically active with the tested substrates and their binding of inhibitors, measured by I50, were not significantly different from the values previously obtained for the wild-type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.     

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.     

Properties of the cysteine residues and the iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Enteromorpha intestinalis

The 5'-adenylyl sulfate (APS) reductase from the marine macrophytic green alga Enteromorpha intestinalis uses reduced glutathione as the electron donor for the reduction of APS to 5'-AMP and sulfite. The E. intestinalis enzyme (EiAPR) is composed of a reductase domain and a glutaredoxin-like C-terminal domain. The enzyme contains a single [4Fe-4S] cluster as its sole prosthetic group. Three of the enzyme's eight cysteine residues (Cys166, Cys257, and Cys260) serve as ligands to the iron-sulfur cluster. Site-directed mutagenesis experiments and resonance Raman spectroscopy are consistent with the presence of a cluster in which only three of the four ligands to the cluster irons contributed by the protein are cysteine residues. Site-directed mutagenesis experiments suggest that the thiol group of Cys250, a residue found only in algal APS reductases, is not an absolute requirement for activity. The other four cysteines that do not serve as cluster ligands, all of which are required for activity, are involved in the formation of two redox-active disulfide/dithiol couples. The couple involving Cys342 and Cys345 has an E(m) value at pH 7.0 of -140 mV, and the one involving Cys165 and Cys285 has an E(m) value at pH 7.0 of -290 mV. The C-terminal portion of EiAPR, expressed separately, exhibits the cystine reductase activity characteristic of glutaredoxins. It is proposed that the Cys342-Cys345 disulfide provides the site for entry of electrons from reduced glutathione and that the Cys166-Cys285 disulfide may serve as a structural element that is essential for keeping the enzyme in the catalytically active conformation.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis

Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.     

Modulation of class Pi glutathione transferase activity by sulfhydryl group modification

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.     

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.     

Cys-X scanning for expansion of active-site residues and modulation of catalytic functions in a glutathione transferase

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.     

Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106)

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.     

Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.     

Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys(95), Cys(82), and Cys(61), and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys(61) generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys(61) as the peroxidatic center. In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.     

Structural determinants of substrate access to the disulfide oxidase Erv2p

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.     

Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

In HspA from Helicobacter pylori vicinal disulfide bridges are a key determinant of domain B structure

Helicobacter pylori produces a heat shock protein A (HspA) that is unique to this bacteria. While the first 91 residues (domain A) of the protein are similar to GroES, the last 26 (domain B) are unique to HspA. Domain B contains eight histidines and four cysteines and was suggested to bind nickel. We have produced HspA and two mutants: Cys94Ala and Cys94Ala/Cys111Ala and identified the disulfide bridge pattern of the protein. We found that the cysteines are engaged in three disulfide bonds: Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 that result in a unique closed loop structure for the domain B.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.