Oxidative folding of lysozyme with aromatic dithiols, and aliphatic and aromatic monothiols

In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox buffers containing asymmetric dithiols 1-5, which possess an aromatic and aliphatic thiol, and symmetric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0 and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathione disulfide, the standard redox buffer, dithiols 1-5 improved the protein folding rates but not the yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8-17 times and improved the yields 15-42% at 1mg/mL lysozyme. Moreover, aromatic dithiol 6 increased the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic dithiols should be useful for protein folding, especially at high protein concentrations.     

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.     

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.     

Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin

The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10muM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb> = 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine. (c) 1994 John Wiley & Sons, Inc.     

DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo

Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.     

Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol pKa, and thiol concentration

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0-8.5) but not at neutral pH values (6-7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10-23 times faster at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol pK(a) values and folding rates at each pH value suggest that the apparent folding rate constants (k(app)) are a function of the redox buffer properties (pH, thiol pK(a) and [RSH]). Fitting the observed data to a three-variable model (logk(app)=-4.216(+/-0.030)+0.5816(+/-0.0036)pH-0.233(+/-0.004)pK(a)+log(1-e(-0.98(+/-0.02)[RSH]))) gave good statistics: r2=0.915, s=0.10.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis.

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30°C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 × 10(-3) s(-1)) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.     

Diselenides as universal oxidative folding catalysts of diverse proteins

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides. Compared to the disulfide-linked dimer of glutathione (the most commonly used oxidant for in vitro protein folding), selenoglutathione provided markedly improved efficiencies in the folding of biotechnologically important proteins such as hirudin, lysozyme, human epidermal growth factor and interferon α-2a. Selenoglutathione also enhances the renaturation of more challenging targets such as bovine serum albumin, whose native state contains 17 disulfide bonds, and the Fab fragment of an antibody. In the latter case, micromolar amounts of selenoglutathione are able to match the modest yield provided by a previously optimized redox buffer, which contains millimolar levels of glutathione. Taken together, the folding reactions of these diverse proteins exemplify the advantages and limitations of diselenide catalysts.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.     

Molecular Mechanisms of Glutaredoxin Enzymes: Versatile Hubs for Thiol–Disulfide Exchange between Protein Thiols and Glutathione

The tripeptide glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) constitute a key redox couple in cells. In particular, they partner protein thiols in reversible thiol–disulfide exchange reactions that act as switches in cell signaling and redox homeostasis. Disruption of these processes may impair cellular redox signal transduction and induce redox misbalances that are linked directly to aging processes and to a range of pathological conditions including cancer, cardiovascular diseases and neurological disorders. Glutaredoxins are a class of GSH-dependent oxidoreductase enzymes that specifically catalyze reversible thiol–disulfide exchange reactions between protein thiols and the abundant thiol pool GSSG/GSH. They protect protein thiols from irreversible oxidation, regulate their activities under a variety of cellular conditions and are key players in cell signaling and redox homeostasis. On the other hand, they may also function as metal-binding proteins with a possible role in the cellular homeostasis and metabolism of essential metals copper and iron. However, the molecular basis and underlying mechanisms of glutaredoxin action remain elusive in many situations. This review focuses specifically on these aspects in the context of recent developments that illuminate some of these uncertainties.     

Role of ascorbate in oxidative protein folding

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.     

Catalysis of oxidative protein folding by small-molecule diselenides

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low p K a of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.     

A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.     

Increased catalytic activity of protein disulfide isomerase using aromatic thiol based redox buffers

PDI is an enzyme that acts as a chaperone, shufflase, and oxidase during the folding of disulfide-containing proteins. The ability of aromatic thiols to increase the activity of PDI-catalyzed protein folding over that of the standard thiol glutathione (GSH) was measured. 4-Mercaptobenzoic acid (ArSH) increased the activity of PDI by a factor of three.     

Oxidative folding of reduced and denatured huwentoxin-I

Huwentoxin-I, a neurotoxic peptide with 33 ammo acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4°C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.     

The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects

There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.     

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.