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PubMed2003

The CXC motif: a functional mimic of protein disulfide isomerase.

Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding. Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI. This peptide has a thiol acid dissociation constant (pK(a) = 8.7) that is lower than that of glutathione. These attributes endow the CGC peptide with substantial disulfide isomerization activity. Escherichia coli thioredoxin (Trx) utilizes the active site sequence Cys-Gly-Pro-Cys (CGPC; E degrees ' = -270 mV) to effect disulfide reduction. Removal of the proline residue from the Trx active site yields a CGC active site with a greatly destabilized disulfide bond (E degrees ' >or= -200 mV). The DeltaP34 variant retains high conformational stability and remains a substrate for thioredoxin reductase. In contrast to the reduced form of the wild-type enzyme, the reduced form of DeltaP34 Trx has disulfide isomerization activity, which is 25-fold greater than that of the CGC peptide. Thus, the rational deletion of an active site residue can bestow a new and desirable function upon an enzyme. Moreover, a CXC motif, in both a peptide and a protein, provides functional mimicry of PDI.

Amino Acid Motifs↗
PubMed1995

DsbA-mediated disulfide bond formation and catalyzed prolyl isomerization in oxidative protein folding.

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1998

Introduction of a disulfide bond into a cationic lipid enhances transgene expression of plasmid DNA.

We have introduced a convenient method of synthesis for disulfide-containing cationic lipids. The lipid, 1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), was synthesized and used to prepare liposomes in combination with DOPE. The rationale behind the selection of the disulfide bond was to produce a lipid which could be selectively destabilized within the cytosol of the cell. The disulfide bond of DOGSDSO was shown to be cleaved by reductive media leading to destabilization of the liposome/DNA complex, thus increasing the release of pDNA compared to a non-disulfide-containing analog. The introduction of a disulfide bond increases the transfection activity using model animal cell lines. The transfection activity and toxicity of DOTAP, DOGSDSO and its analog in three cell lines were compared. The amount of transgene (e.g. luciferase) produced with the use of DOGSDSO/DOPE was greater than that of DOTAP/DOPE and up to 50 times more than that of its non-disulfide analog. The results indicate disulfide-containing cationic liposomes may act as excellent vectors for gene transfection.

Cations↗
PubMed1984

Thiol-disulfide interchange between cystine and N-2-mercaptoethyl-1, 3-diaminopropane as a potential treatment for cystinuria.

The radioprotective compound WR2721 is a thiophosphate, which, when administered orally, is activated at the acid pH of the stomach to its free thiol (MDP). The free thiol is a mucolytic compound which acts via the reduction of disulfide bonds of mucin molecules. An equimolar mixture of MDP and cysteine, in urine at pH 6.0 and 37 degrees C, when oxidized by molecular oxygen, preferentially forms the soluble mixed disulfide between MDP and cysteine. The disulfide cystine will undergo thiol-disulfide interchange with MDP; as a result, cystine crystals are effectively dissolved. Moreover, in the presence of catalytic amounts of free thiol, the disulfide of MDP will undergo thiol-disulfide interchange with cystine to dissolve cystine crystals. The mixed disulfide of MDP with cysteine is soluble in urine at pH 6.0 and 37 degrees C to at least 100 mg/ml. Chromatographic procedures which permit the analysis of MDP and its mixed disulfide derivatives as MDP-sulfonic acid are described. By these procedures, it was demonstrated that 20% of a single oral dose of WR2721 was excreted as MDP derivatives in the urine of normal volunteers. These procedures will permit the evaluation of WR2721 in the treatment of cystinuria.

Amifostine↗
PubMed2000

Copper(II) and nickel(II) complexes of binucleating macrocyclic bis(disulfide)tetramine ligands.

Novel macrocyclic bis(disulfide)tetramine ligands and several Cu(II) and Ni(II) complexes of them with additional ligands have been synthesized by the oxidative coupling of linear tetradentate N2S2 tetramines with iodine. Facile demetalation of the Ni(II) oxidation products affords the free 20-membered macrocycles meso-9 and rac-9 and the 22-membered macrocycle 16, all of which are potentially octadentate N4S4 ligands. X-ray structure analyses reveal distinctly different conformations for the two isomers of 9; meso-9 shows a stepped conformation in profile with the disulfide groups corresponding to the rise of the step, whereas rac-9 exhibits a V conformation with the disulfide groups near the vertex of the V. No metal complexes of rac-9 have been isolated. Crystallographic studies of three Cu(II) complexes reveal that depending upon the size of the macrocyclic ligand and the nature of the additional ligands (I-, NCO-, and CH3CN), the Cu(II) coordination geometry shows considerable variation (plasticity), with substantial changes in the Cu(II)-disulfide bonding. Thus, a diiodide salt contains six-coordinate Cu(II) to which all four bridging disulfide sulfur atoms form strong equatorial bonds. In contrast, isocyanato complexes of the 20- and 22-membered macrocycles exhibit trigonal-bipyramidal Cu(II) and distorted cis-octahedral Cu(II) geometries, respectively, having only one and no short equatorially bound sulfur atoms. The coordination geometry of the latter complex can also be described as four-coordinate seesaw with two semicoordinated S(disulfide) ligands. Disulfide-->Cu(II) ligand-to-metal charge transfer absorptions of both isocyanato-containing Cu(II) species appear too weak to observe, probably because of poor overlap of the sulfur orbitals with the Cu(II) d-vacancy. The dual disulfide-bridged Ni(II) units of the crystallographically characterized octahedral Ni(II) complex of meso-9 with axial iodide and acetonitrile ligands promote substantial antiferromagnetic coupling (J = -13.0(2) cm-1).

Journal Article↗
PubMed2000

Conformational preferences and supramolecular aggregation in 2-nitrophenylthiolates: disulfides and thiosulfonates.

In each of the asymmetrically substituted disulfides 2-nitro-4'-methyldiphenyl disulfide, C(13)H(11)NO(2)S(2) (1), 2-nitro-4'-chlorodiphenyl disulfide, C(12)H(8)ClNO(2)S(2) (2), 2, 4-dinitro-4'-methyldiphenyl disulfide, C(13)H(10)N(2)O(4)S(2) (3), and 2,4-dinitrophenyl-2'-methoxycarbonylmethyl disulfide, C(9)H(8)N(2)O(6)S(2) (4), and in both of the symmetrically substituted disulfides bis(2-nitrophenyl) disulfide, C(12)H(8)N(2)O(4)S(2) (5), and bis(2-nitro-4-trifluoromethylphenyl) disulfide, C(14)H(6)F(6)N(2)O(4)S(2) (6), the 2-nitro groups are essentially coplanar with the adjacent aryl ring and the S atom remote from the nitrated aryl ring is also essentially coplanar and transoid to the nitro group. In S-(2-nitrophenyl) 2-nitrobenzene thiosulfonate, C(12)H(8)N(2)O(6)S(2) (7), which contains three independent molecules in the asymmetric unit, all six of the independent nitro groups are twisted out of the plane of the adjacent aryl rings. The crystal structures of (1)-(3) contain isolated molecules, that of (4) contains centrosymmetric dimers held together by C-H.O hydrogen bonds, while in the structures of (5)-(7), respectively, the C-H.O hydrogen bonds generate one-, two- and three-dimensional arrays. The interplay between molecular conformation and supramolecular aggregation is discussed.

Journal Article↗
PubMed2003

Role of disulfide bonds in the structure and activity of human insulin.

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Analysis of disulfide-linked homo- and hetero-peptide dimers with a strong cation-exchange sulfoethyl aspartamide column.

A strong cation-exchange sulfoethyl aspartamide column was used to analyze and purify disulfide-linked homo-/hetero-peptide dimers. Monomeric peptides elute from this column in a monotonic fashion according to their net nominal positive charge at pH 3.0. Disulfide-linked peptide dimers are expected to possess an increased net positive charge at pH 3.0 and therefore should elute later and be well resolved from their monomeric constituents. Five distinct synthetic peptides (7 to 14 residues in length) ranging in net nominal charge at pH 3.0 from +1 to +4, with cysteine located at the N-terminus, C-terminus, or at an internal position, were used to produce disulfide-linked homo/hetero-peptide dimers as follows: The peptide was first reacted with 5,5'-dithiobis (2-nitrobenzoic acid) to form the mixed-disulfide, peptide:2-nitro-5-thiobenzoic acid adduct which is easily monitored at 325 nm. Then, the second cysteine-containing peptide was added and the desired disulfide-linked homo-/hetero-peptide dimer was produced via a thiol-disulfide interchange reaction. The entire reaction mixture was subsequently chromatographed on the sulfoethyl aspartamide column to isolate the disulfide-bonded species and also, to identify other reaction products. In addition, the same reaction mixture was analyzed by standard C18 reverse-phase chromatography to compare the capabilities of these two distinct chromatographic modes to identify disulfide-linked homo-/hetero- peptide dimers. It is shown that each chromatographic system successfully resolved all five homo-peptide dimers from their respective monomer counterparts, with separation being slightly better on the sulfoethyl aspartamide column.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Size-exclusion high performance liquid chromatography of native trypsinogen, the denatured protein, and partially refolded molecules. Further evidence that non-native disulfide bonds are dominant in refolding the completely reduced protein.

Size-exclusion high performance liquid chromatography was used to compare the Stokes radius of the mixed disulfide of trypsinogen refolded for 10 min with the Stokes radius of denatured trypsinogen in high concentrations of urea. After folding for 10 min, rechromatography of a collection of sequential fractions of an initial separation showed that the fractions display microheterogeneity as seen in the value of the Stokes radius of each fraction. These intermediate species differed in their Stokes radius, and each had a globular structure cross-linked by disulfide bonds. In contrast, when trypsinogen with the native disulfides intact was equilibrated at different concentrations of urea (0-8 M), a progressive increase in Stokes radius was observed with extent of unfolding. Rechromatography of a series of fractions collected at a specific urea concentration showed that each had the same Stokes radius as the fraction in the initial separation. Urea-denatured trypsinogen and partially refolded trypsinogen must therefore differ in the disulfide pairing that links regions of the polypeptide chain. These observations support the suggestion that non-native disulfide bonds are responsible for the many stable conformations that form early in the folding of the mixed disulfide of trypsinogen (Light, A., and Higaki, J.N. (1987) Biochemistry 26, 5556-5564). These intermediates initially are loose structures (large Stokes radius) that become more compact with time (decreasing Stokes radius). The intermediates must therefore undergo a continuing disulfide interchange until native disulfides form late in the process when the stable conformation of the native molecule is reached.

Chromatography, High Pressure Liquid↗
PubMed1993

Determination and metabolism of dithiol chelating agents. XV. The meso-2,3-dimercaptosuccinic acid-cysteine (1:2) mixed disulfide, a major urinary metabolite of DMSA in the human, increases the urinary excretion of lead in the rat.

Meso-2,3-dimercaptosuccinic acid (DMSA) in humans is an effective p.o. therapeutically useful chelating agent of Pb. In humans given DMSA p.o., the major urinary metabolite is DMSA-cysteine (1:2) mixed disulfide. In order to determine its efficacy in mobilizing Pb and increasing urinary Pb excretion, the mixed disulfide was given to rats treated previously with Pb acetate. The mixed disulfide was as effective as DMSA in increasing the urinary excretion of Pb and mobilizing Pb from the kidney. DMSA, however, appears to be superior for mobilizing Pb from the liver and the brain. After the mixed disulfide was given s.c. to rats, DMSA was found in the blood and urine. Twenty-four hours after administration, 0.7% of the administered mixed disulfide was found in the urine as DMSA, indicating the mixed disulfide can be reduced to DMSA. The mixed disulfide was also reduced in vitro to DMSA during incubation with rat blood. Although in the rat the DMSA-cysteine (1:2) mixed disulfide mobilized Pb from the kidney, increased the urinary excretion of Pb and was to some extent reduced to DMSA, its fate and pharmacological properties in the human, where it is found after DMSA administration, are unknown.

Animals↗
PubMed1998

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.

Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.

Animals↗
PubMed1996

Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine.

Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.

Amino Acid Sequence↗
PubMed1996

Folding of omega-conotoxins. 1. Efficient disulfide-coupled folding of mature sequences in vitro.

Disulfide-coupled refolding reactions of five omega-conotoxins, Ca2+ channel antagonists derived from marine snails of the genus Conus, were examined. These peptides are 23-26 amino acid residues long, and the native conformation of each is stabilized by three disulfide bonds. Although the primary structures of the peptides show only limited sequence similarity, the patterns of disulfides and three-dimensional conformations are very similar. Refolding of the reduced proteins was promoted by the disulfide form of glutathione (GSSG) in the presence of reduced glutathione (GSH). All five of the peptides examined were able to refold to the native conformation, as judged by reversed-phase HPLC behavior, with efficiencies of 16% for omega-MVIIC, 28% for omega-MVIID, and 50% for omega-MVIIA, omega-GVIA, and omega-SVIA. The refolded form of omega-MVIIA was further shown to have biological activity indistinguishable from that of the native form, as well as the same rate of reductive unfolding in the presence of dithiothreitol. The overall folding rate and efficiency of omega-MVIIA was found to be quite sensitive to the thiol-disulfide redox potential, with optimum rates and yields obtained in the presence of GSSG and GSH at concentrations similar to those believed to be present in the endoplasmic reticulum. The folding efficiency of this peptide was greatly reduced by the addition of 8 M urea, indicating that formation of the correct disulfides is determined largely by noncovalent interactions, as opposed to steric constraints arising from the spacing between Cys residues. These results demonstrate that the mature forms of at least some omega-conotoxins contain sufficient information to direct correct folding and disulfide formation, in spite of their small size and limited sequence conservation.

Amino Acid Sequence↗
PubMed1996

Rapid formation of the native 14-38 disulfide bond in the early stages of BPTI folding.

Using recombinant variants of BPTI, we have determined the rate constants corresponding to formation of each of the fifteen possible disulfide bonds in BPTI, starting from the reduced, unfolded protein. The 14-38 disulfide forms faster than any of the other 14 possible disulfides. This faster rate results from significantly higher intrinsic chemical reactivities of Cys-14 and Cys-38, in addition to local structure in the reduced protein that facilitates formation of the 14-38 disulfide bond. This disulfide bond is found in native BPTI. Our results suggest that a significant flux of folding BPTI molecules proceed through the one-disulfide intermediate with the 14-38 disulfide bond, denoted [14-38], that has recently been detected on the BPTI folding pathway. In addition to providing a detailed picture of the early events in the folding of BPTI, our results address quantitatively the effect of local structure in the unfolded state on folding kinetics.

Amino Acid Sequence↗
PubMed1997

Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from Escherichia coli.

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and required for efficient disulfide bond formation in the bacterial periplasm. The enzyme consists of a thioredoxin-like domain and a second, alpha-helical domain which is inserted into the thioredoxin motif. Reduction of the active-site disulfide in the thioredoxin domain causes a more than 3-fold increase in tryptophan fluorescence. However, both tryptophan residues of the protein, W76 and W126, are not in contact with the disulfide and located in the alpha-helical domain. Analysis of the variants W76F and W126F revealed that the fluorescence of W126 is fully quenched in every redox state of DsbA. W126 is also a sink for nonradiative energy transfer from W76. In oxidized DsbA, W76 is quenched by an intramolecular, dynamic quenching process which involves energy transfer from W76 via F26 to the disulfide. The contributions of the disulfide bridge and the tryptophan residues to the near-UV CD spectra were also quantified. Analysis of the thermodynamic stabilities of the variants W76F and F26L revealed that the interdomain contact between W76 and F26 strongly contributes to the overall stability of DsbA, and selectively stabilizes its oxidized form. The DsbA variant F26L is the most oxidizing disulfide oxidoreductase known so far.

Bacterial Proteins↗
PubMed1997

Regeneration studies of an analog of ribonuclease A missing disulfide bonds 65-72 and 40-95.

Mutants of bovine pancreatic ribonuclease A (RNase A) that contain four of the eight cysteine residues found in the wild-type protein were prepared. Cysteine residues 40, 65, 72, and 95 were replaced by serine to form [C40S,C65S,C72S,C95S] RNase A or by alanine to form [C40A,C65A,C72A,C95A] RNase A, which contain the following four cysteine residues: 26, 58, 84, and 110. The substitutions resulted in deletion of wild-type disulfide bonds, 65-72 and 40-95. These mutants were prepared to investigate interactions that may be important for the folding and unfolding of the wild-type protein. The mutant protein was expressed and purified in an unfolded sulfonated form. Upon regeneration of the native form from the reduced mutant with DTTox, all three of the possible two-disulfide pairings, including the native one, formed. One-dimensional 1H NMR spectra demonstrated that the conformations of these three species are similar and are predominantly disordered; however, there is evidence of local structure in the vicinity of one histidine residue. It was also shown that disulfide pairing is not completely random and that both entropic factors and enthalpic interactions contribute to the formation of the native-disulfide bonds. The presence of more than a statistical population of native-disulfide pairings indicates that specific local interactions present in the reduced protein direct the preferential formation of native-disulfide bonds in the two-disulfide mutant.

Animals↗
PubMed1998

Conversion of a catalytic into a structural disulfide bond by circular permutation.

The thiol-disulfide oxidoreductase DsbA from Escherichia coli is the strongest oxidant of the enzyme family and required for disulfide bond formation in the bacterial periplasm. The catalytic domain of this 189-residue protein has a thioredoxin-like fold and contains a catalytic disulfide bridge that is located within the sequence Cys30-Pro31-His32-Cys33 at the N-terminus of an alpha-helix. The Cys30-Cys33 disulfide bond destabilizes DsbA by about 16 kJ/mol at pH 7.0, which appears to be caused by the extremely low pKa value of approximately 3.4 of the nucleophilic Cys30 thiol. Here we report the characterization of a circularly permuted variant of DsbA, termed H32-P31, in which the natural termini are connected by a Gly3-Thr-Gly linker and the new termini are located between the active-site cysteines (first residue His32, last residue Pro31). The disulfide bond in the variant thus connects the second with the penultimate residue. H32-P31 adopts a wild-type-like structure and folds reversibly and cooperatively in both redox forms. However, the permuted variant is catalytically inactive as dithiol oxidase in vivo and in vitro. Both cysteine thiols have pKa values > 8; the variant is 500-fold more reducing than the wild type and more stable in its oxidized form. Thus, the Cys30-Cys33 disulfide in the variant H32-P31 has adopted properties of a structural disulfide bond.

Catalysis↗
PubMed1993

Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase.

Evidence had been provided that a disulfide-linked [125I]iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thiol-disulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI--in particular, bacitracin and anti-PDI antibodies--might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties.

4-Chloromercuribenzenesulfonate↗