E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

The redox‐sensing regulator YodB senses quinones and diamide via a thiol‐disulfide switch in Bacillus subtilis

The MarR/DUF24‐type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox‐sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6‐Ala mutant that the conserved Cys6 apparently contributes to the DNA‐binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox‐sensing in response to diamide and 2‐methylhydroquinone (MHQ) in vivo. DNA‐binding experiments indicate that YodB is regulated by a reversible thiol‐modification in response to diamide and MHQ in vitro. Redox‐regulation of YodB involves Cys6‐Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C‐terminal Cys101' or Cys108' residues. This study reveals a thiol‐disulfide switch model of redox‐regulation for the YodB repressor to sense electrophilic compounds in vivo.     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

The involvement of catalytic site thiol groups in the activation of soluble guanylate cyclase by sodium nitroprusside

Sodium nitroprusside, a potent activator of soluble guanylate cyclase, potentiated mixed disulfide formation between cystine, a potent inhibitor of the cyclase, and enzyme purified from rat lung. Incubation of soluble guanylate cyclase with nitroprusside and [35S]cystine resulted in a twofold increase in protein-bound radioactivity compared to incubations in the absence of nitroprusside. Purified enzyme preincubated with nitroprusside and then gel filtered (activated enzyme) was activated 10- to 20-fold compared to guanylate cyclase preincubated in the absence of nitroprusside and similarly processed (nonactivated enzyme). This activation was completely reversed by subsequent incubation at 37 degrees C (activation-reversed enzyme). Incorporation of [35S]cystine into guanylate cyclase was increased twofold with activated enzyme, while no difference was observed with activation-reversed enzyme, compared to nonactivated enzyme. Cystine decreased the activity of nonactivated and activation-reversed enzyme about 40% while it completely inhibited activated guanylate cyclase. Mg+2- or Mn+2-GTP inhibited the incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. Also, diamide, a potent thiol oxidant that converts juxtaposed sulfhydryls to disulfides, completely blocked incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. These data indicate that activation of soluble guanylate cyclase by nitroprusside results in an increased availability of protein sulfhydryl groups for mixed disulfide formation with cystine. Protection against mixed disulfide formation with diamide or substrate suggests that these groups exist as two or more juxtaposed sulfhydryl groups at the active site or a site on the enzyme that regulates catalytic activity. Differential inhibition by mixed disulfide formation of nonactivated and activated enzyme suggests a mechanism for amplification of the on-off signal for soluble guanylate cyclase within cells.     

Dopamine biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylast inhibition during oxidative stress

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inhibited by the sulfhydryl oxidant diamide in a concentration-dependent manner. The inhibitory effect of diamide on TH catalytic activity is enhanced significantly by GSH. Treatment of TH with diamide in the presence of [(35)S]GSH results in the incorporation of (35)S into the enzyme. The effect of diamide-GSH on TH activity is prevented by dithiothreitol (DTT), as is the binding of [(35)S]GSH, indicating the formation of a disulfide linkage between GSH and TH protein cysteinyls. Loss of TH catalytic activity caused by diamide-GSH is partially recovered by DTT and glutaredoxin, whereas the disulfide linkage of GSH with TH is completely reversed by both. Treatment of intact PC12 cells with diamide results in a concentration-dependent inhibition of TH activity. Incubation of cells with [(35)S]cysteine, to label cellular GSH prior to diamide treatment, followed by immunoprecipitation of TH shows that the loss of TH catalytic activity is associated with a DTT-reversible incorporation of [(35)S]GSH into the enzyme. A combination of matrix-assisted laser desorption/ionization/mass spectrometry and liquid chromatography/tandem mass spectrometry was used to identify the sites of S-glutathionylation in TH. Six cysteines (177, 249, 263, 329, 330, and 380) of the seven cysteine residues in TH were confirmed as substrates for modification. Only Cys-311 was not S-glutathionylated. These results establish that TH activity is influenced in a reversible manner by S-glutathionylation and suggest that cellular GSH may regulate dopamine biosynthesis under conditions of oxidative stress or drug-induced toxicity.     

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure/function of human type 1 3beta-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.     

Yeast thioltransferase--the active site cysteines display differential reactivity

Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae. The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography. Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography. The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system. Both proteins showed similar amino acid compositions and identical N-termini. The major peak of thioltransferase was extensively characterized. Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics. The enzyme activity had a pH optimum of 9.1. The protein has 106 amino acid residues with two cysteines and no arginine. The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin. The two cysteines at the active site displayed different reactivities to iodoacetamide. Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0. The enzyme was completely inactivated when the Cys26 was carboxymethylated. A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity. The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Activation of glucuronidation through reduction of a disulfide bond in rat UDP-glucuronosyltransferase 1A6

UDP-glucuronosyltransferase- (UGT-) dependent glucuronidation is an important detoxification process for many endogenous and exogenous compounds in mammals. Treatment of rat hepatic microsomes with the reducing reagent dithiothreitol (DTT) resulted in a significant increase in p-nitrophenol (p-NP) glucuronidation in a time- and concentration-dependent manner. The DTT-dependent activation of glucuronidation was specific for planar phenols but not for bilirubin or testosterone without membrane perturbation of the microsomes. p-NP glucuronidation in Gunn rat hepatic microsomes lacking UGT1 isozymes was not affected by DTT, indicating that UGT1A6 in the microsomes is mainly involved in the activation. The DTT-dependent activation was inhibited by 1,6-bis(maleimido)hexane (BMH) but not by N-ethylmaleimide, indicating that cross-linking between cysteine residues in UGT1A6 is responsible for the activation. Immunoblot analysis of rat hepatic microsomes on nonreducing SDS-PAGE gels revealed that most of the UGT1A6 migrated as a monomer, suggesting that DTT could affect an intramolecular disulfide bond in the UGT1A6 that may be responsible for the activation. To identify which of the ten cysteines in UGT1A6 are involved in the disulfide bond, rat UGT1A6 wild type and a set of mutants, each with a cysteine to serine substitution, were constructed and expressed in COS cells. Treatment of COS microsomes with DTT had no effect on the activity of the wild type but BMH showed significant inhibition, suggesting that UGT1A6 expressed in COS cells may be in the reduced and activated state. Replacement of either Cys 121 or Cys 125 with serine showed insensitivity to the BMH-dependent inhibition. These results demonstrate that both Cys 121 and Cys 125 are responsible for the activation of the activity through the disulfide bond in rat UGT1A6.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

Investigation of the oligomeric status of the peroxisomal isoform of human 3-hydroxy-3-methylglutaryl-CoA lyase

Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity. The C323S mutant shows little DTT activation. Superose gel filtration chromatography data have prompted other investigators to hypothesize that the peroxisomal isoform is a monomer. This study confirms the elution properties presented in that earlier report, but also demonstrates anomalous elution up on Superose chromatography. Elution properties observed using a polyacrylamide resin (Bio-Gel P100) suggest a dimeric, rather than monomeric, enzyme. This observation has been further tested by protein chemistry experiments. The peroxisomal enzyme forms a covalently linked dimeric species upon crosslinking with dibromopropanone or o-phenylenedimaleimide or upon disulfide formation as a result of incubation with diamide. Cysteine-323 is required for intersubunit covalent crosslinking. Crosslinking efficiency is not dependent on HMG-CoA lyase protein concentration nor is it influenced by the presence of varying concentrations of an unrelated protein, such as ovalbumin. Sedimentation equilibrium analyses do not indicate a monomeric form of either human mitochondrial or human peroxisomal HMG-CoA lyase; the results suggest that these proteins are predominantly dimers. The retention of the basic N-terminal mitochondrial signal sequence in the peroxisomal HMG-CoA lyase isoform may influence elution from Superose gel filtration media but does not alter the oligomeric status of the enzyme.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Influence of the activation status and of ATP on phosphoribulokinase degradation

The light-regulated chloroplast enzyme phosphoribulokinase (EC 2.7.1. 19) exists in two forms. In darkness this enzyme is present in an oxidized form, which is inactive. It is activated in the light by a thioredoxin-mediated reduction. In extracts from young wheat leaves (Triticum aeestivum L.) phosphoribulokinase as well as some other thioredoxin-modulated enzymes can be activated by the artificial reductant dithiothreitol (DTT). The influence of the activation status and of the substrate ATP on phosphoribulokinase stability was investigated in the presence of endogenous endopeptidases from senescing wheat leaves. Similar experiments were performed with purified phosphoribulokinase from spinach in the presence of exogenous, purified endopeptidases (chymotrypsin and trypsin). Phosphoribulokinase stability was analysed by immunoblotting and activity measurements. Both systems led to similar conclusions. DTT (reductant and ATP (substrate) stabilized phosphoribulokinase in wheat leaf extracts as well as partially purified phosphoribulokinase from spinach. The combination of both effectors was far more protective than either effector alone. DTT had hardly any effect on the degradation of thioredoxin-independent chloroplast enzymes such as glutamate synthase and glutamine synthetase. These results suggest that the activation status and substrate concentrations are not only important for the activity of phosphoribulokinase, but are also relevant for the susceptibility of this enzyme to proteolysis.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.