A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Activation of the heterodimeric central complex SoxYZ of chemotrophic sulfur oxidation is linked to a conformational change and SoxY-Y interprotein disulfide formation

The central protein of the four component sulfur oxidizing (Sox) enzyme system of Paracoccus pantotrophus, SoxYZ, carries at the SoxY subunit the covalently bound sulfur substrate which the other three proteins bind, oxidize, and release as sulfate. SoxYZ of different preparations resulted in different specific thiosulfate-oxidizing activities of the reconstituted Sox enzyme system. From these preparations SoxYZ was activated up to 24-fold by different reductants with disodium sulfide being the most effective and yielded a uniform specific activity of the Sox system. The activation comprised the activities with hydrogen sulfide, thiosulfate, and sulfite. Sulfide-activation decreased the predominant beta-sheet character of SoxYZ by 4%, which caused a change in its conformation as determined by infrared spectroscopy. Activation of SoxYZ by sulfide exposed the thiol of the C-terminal Cys-138 of SoxY as evident from alkylation by 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Also, SoxYZ activation enhanced the formation of the Sox(YZ)2 heterotetramer as evident from density gradient gel electrophoresis. The tetramer was formed due to an interprotein disulfide between SoxY to yield a SoxY-Y dimer as determined by combined high pressure liquid chromatography and mass spectrometry. The significance of the conformational change of SoxYZ and the interprotein disulfide between SoxY-Y is discussed.     

An atypical catalytic mechanism involving three cysteines of thioredoxin

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys(4)) and two (Cys(58) and Cys(61)) in the classical thioredoxin active site ((57)WCGPC(61)). The property of a mutant in which Cys(58) was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys(4) is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys(58) can be formed, linking it to either Cys(61) or Cys(4). We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys(4) to convert this atypical thioredoxin h back to its active reduced form.     

Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.     

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.     

Reaction of human myoglobin and H2O2. Electron transfer between tyrosine 103 phenoxyl radical and cysteine 110 yields a protein-thiyl radical

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys(110)). Adding hydrogen peroxide (H(2)O(2)) to human Mb affords Trp(14)-peroxyl, Tyr(103)-phenoxyl, and Cys(110)-thiyl radicals and coupling of Cys(110)-thiyl radicals yields a homodimer through intermolecular disulfide bond formation (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) J. Biol. Chem. 275, 20391-20398). Treating a solution of wild type Mb and H(2)O(2) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein /= 100 mol/mol only DMPO-Tyr(103) radicals were present. The DMPO-dependent decrease in DMPO-Cys(110) was matched by a near 1:1 stoichiometric increase in DMPO-Tyr(103). In contrast, reaction of the Y103F human Mb with H(2)O(2) gave no DMPO-Cys(110) at DMPO:protein /= 100 mol/mol (i.e. conditions that consistently gave DMPO-Tyr(103) in the case of wild type Mb). No detectable homodimer was formed by incubation of the Y103F variant with H(2)O(2). However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H(2)O(2) (C110A:Y103F:H(2)O(2) 2:1:5 mol/mol/mol); the yield of this homodimer increased with increasing ratios of C110A:Y103F. Together, these data suggest that addition of H(2)O(2) to human Mb can produce Cys(110)-thiyl radicals through an intermolecular electron transfer reaction from Cys(110) to a Tyr(103)-phenoxyl radical.     

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.     

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.     

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.     

Insights into structure and function of the active site of SoxAX cytochromes

SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met(236), representing an unusual rotamer of methionine. The flexibility of the SnSoxAX(C236M) SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, -479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s(-1); C236M, 2.0 s(-1)). SnSoxAX(C236M) was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys(236) aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.     

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG beta-subunit residues, termed the seatbelt, are wrapped around alpha-subunit loop 2 (alpha 2) and their positions "latched" by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the beta-subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta-subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require alpha 2 and its attached oligosaccharide to be threaded through a small beta-subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1--3 mM beta-mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93--Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the beta-subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha 2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme. Mutants of human RNase H1 in which the cysteines were either deleted or substituted with alanine exhibited cleavage rates comparable with the reduced form of the enzyme, suggesting that the cysteine residues were not required for catalysis. The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148). The redox states of the Cys(147) and Cys(148) residues were determined by digesting the reduced, oxidized, and NEM-treated forms of human RNase H1 with trypsin and analyzing the cysteine containing tryptic fragments by micro high performance liquid chromatography-electrospray ionization-Fourier transform ion cyclotron mass spectrometry. The tryptic fragment Asp(131)-Arg(153) containing Cys(147) and Cys(148) was identified. The mass spectra for the Asp(131)-Arg(153) peptides from the oxidized and reduced forms of human RNase H1 in the presence and absence of NEM showed peptide masses consistent with the formation of a disulfide bond between Cys(147) and Cys(148). These data show that the formation of a disulfide bond between adjacent Cys(147) and Cys(148) residues results in an inactive enzyme conformation and provides further insights into the interaction between human RNase H1 and the heteroduplex substrate.     

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.     

Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Properties and significance of apoFNR as a second form of air-inactivated [4Fe-4S].FNR of Escherichia coli

The active form of the oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains a [4Fe-4S] cluster which is converted to a [2Fe-2S] cluster after reaction with air, resulting in inactivation of FNR. Reaction of reconstituted [4Fe-4S].FNR with air resulted within 5 min in conversion to apoFNR. The rate was comparable to the rate known for [4Fe-4S].FNR/[2Fe-2S].FNR cluster conversion, suggesting that apoFNR is a product of [2Fe-2S].FNR decomposition and a final form of air-inactivated FNR in vitro. Formation of apoFNR and the redox state of the cysteinyl residues were determined in vitro by alkylation. FNR contains five cysteinyl residues, four of which (Cys20, Cys23, Cys29 and Cys122) ligate the FeS clusters. Alkylated FNR and proteolytic fragments thereof were analyzed by MALDI-TOF. ApoFNR formed by air inactivation of [4Fe-4S].FNR in vitro contained one or two disulfides. Only disulfide pairs Cys16/20 and Cys23/29 were formed; Cys122 was never part of a disulfide. The same type of disulfide was found in apoFNR obtained during isolation of FNR, suggesting that cysteine disulfide formation follows a fixed pattern. ApoFNR, including the form with two disulfides, can be reconstituted to [4Fe-4S].FNR after disulfide reduction. The experiments suggest that apoFNR is a major form of FNR under oxic conditions.     

Evidence for the participation of Cys558 and Cys559 at the active site of mercuric reductase

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH2 form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH2 has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH2 actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 degrees C and at 25 degrees C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog, S. (1983) Biochem. Biophys. Res. Commun. 117, 231-237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured Eox and EH2 enzyme forms show that an additional disulfide is the group in communication with the active site. [14C]Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys558 and Cys559, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys558 and Cys559 as thiols exhibits less than 50% of the fluorescence of Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.     

Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.     

An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys(61) and Cys(177) produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.