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PubMed2005

Synthesis, reactivity and structural studies of carboranyl thioethers and disulfides.

The equimolar reaction of 1-SH-2-R-1,2-closo-C2B10H10(R=Me, H, Ph) with KOH in ethanol produces the thiolate species [1-S-2-R-1,2-closo-C2B10H10]-. These react with iodine to give the disulfide bridged dicluster (1-S-2-R-1,2-closo-C2B10H10)2(R=H, Me, Ph) compounds as analytically pure, white and air-stable solids in high yield. Synthesis of monothioether bridged species is synthetically more difficult. In fact three procedures have been tested to obtain the thioether bridged dicluster compounds (2-R-1,2-closo-C2B10H10)2S (R=Me, H, Ph) but only (2-Me-1,2-closo-C2B10H10)2S was successfully synthesized and characterized. Attempts to produce mixed compounds (1-R-1,2-closo-C2B10H10)S(1-R'-1,2-closo-C2B10H10), R not=R', were unsuccessful. Deboronation reaction of this dicarboranylthioether lead, depending on the reaction conditions, to monoanionic [(2-Me-1,2-closo-C2B10H10)S(8-Me-7,8-nido-C2B9H10)]- or dianionic [(8-Me-7,8-nido-C2B9H10)2S]2- sulfur bridge anions. Deboronation of carboranyl disulfides gave the corresponding dianionic [(7-S-8-R-7,8-nido-C2B9H10)2]2-(R=H, Me, Ph) species. This reaction was very dependent, however, on the reaction conditions. With slight variation of the reaction conditions, splitting of the S-S bond leading to the thiolate species with retention of the closo cluster was also found. Carboranyl disulfides (1-S-2-R-1,2-closo-C2B10H10)2(R=H, Me, Ph) do not lead to thiosulfinates R-S(O)-S-R' by oxidation with H2O2 or I2 as organic disulfides do. This behaviour is attributed to the presence of the sulfur atom directly bonded to the carbon cluster that produces electronic transfer from the filled orbitals on the sulfur atom into the cage LUMO (largely located on the cage Cc-Cc bond). This causes a depletion of electron density on the sulfur, thence impairing sulfur oxidation, and facilitating S-S breaking. Crystal structures of monothioethers (2-Me-1,2-closo-C2B10H10)2S, [NMe4][(2-Me-1,2-closo-C2B10H10)S(8-Me-7,8-nido-C2B9H10)](the first example reported in the literature of a two cluster compound incorporating the closo C2B10 and the nido[C2B9]- moieties linked by a one member spacer) and disulfides (1-S-1,2-closo-C2B10H11)2, (1-S-2-Me-1,2-closo-C2B10H10)2, (1-S-2-Ph-1,2-closo-C2B10H10)2 are reported which support the behaviour of these species.

Journal Article↗
PubMed2001

Maturation of Pseudomonas aeruginosa elastase. Formation of the disulfide bonds.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.

Bacterial Proteins↗
PubMed1996

Peroxynitrite modification of glutathione reductase: modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide binding site.

The catalytic properties of glutathione reductase for its substrate, glutathione disulfide, were altered following a 60 s exposure to a 100-fold molar excess of peroxynitrite; the K(M) value was increased by approximately 2.5-fold and the V(max) value was decreased by approximately 1.7-fold. The kinetic alterations are thought to result from nitrotyrosine formation as the intrinsic Tyr fluorescence is diminished. The UV-visible spectrum of glutathione reductase exhibited absorbance at approximately 423 nm, characteristic of nitrotyrosine. In addition, the presence of nitrotyrosine has been detected by Western immunoblots with an anti-nitrotyrosine antibody. The peroxynitrite-induced inactivation is not observed in the presence of excess glutathione disulfide. However, excess NADPH offered no protection against peroxynitrite-induced inactivation. These observations suggest that the modification of approximately 1.8 Tyr per subunit, at or near the glutathione disulfide binding domain, probably results in the observed catalytic alterations. To test this hypothesis, the two tyrosines closest to the glutathione disulfide binding domain (Tyr114 and Tyr106), as indicated by the X-ray crystallographic data [Karplus and Schulz (1989) J. Biol. Chem., 210, 163-180], were each converted to nitrotyrosines by molecular modeling and the structure energy was minimized. These theoretical calculations indicate that the bond lengths between Tyr114-O and the Gly-N and Cys II-N of glutathione disulfide bound to glutathione reductase (Karplus and Schulz, 1989) increased by 3.0 and 4.3 A, respectively, upon nitration. In the case of Tyr106 the 0-Cys II-N distance also increases by approximately 1.6 A. The loss of these hydrogen bonding contacts is likely to result in the observed catalytic alterations upon reaction with peroxynitrite.

Animals↗
PubMed2003

Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding.

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.

Animals↗
PubMed1988

New segment synthesis of alpha-inhibin-92 by the acyl disulfide method.

The thiocarboxyl group reacts with diaryl disulfides to give an unsymmetrical acyl disulfide in dimethylformamide (DMF) and a symmetrical diacyl disulfide in aqueous DMF. Both acyl disulfides react with the alpha-amino group to form the peptide bond. The method was used in a new segment synthesis of alpha-inhibin-92 (alpha-IB-92) with use of 2,2'-dipyridyl disulfide as activator. Thiocarboxyl peptides were synthesized by the solid-phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethyl-resin. The segments alpha-IB-92-(1-34)SH (I), Msc-alpha-IB-92-(35-65)SH (II), Msc-alpha-IB-92(66-92)OH (III), and Msc-alpha-IB-92-(35-92)OH (VI) were prepared in yields of 33, 36, 41, and 25%, respectively, with use of crystalline symmetrical anhydrides in double and triple coupling protocols. Segments I, II, and III were used in a 3-segment synthesis of alpha-IB-92 with an overall yield based on starting resin of about 8% while a 2-segment synthesis using I and IV gave 11%. An all stepwise synthesis of alpha-IB-92 gave 4.5%.

Amino Acid Sequence↗
PubMed2005

Oxidative folding of conotoxins sharing an identical disulfide bridging framework.

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.

Animals↗
PubMed1992

Contribution of disulfide bonds in the carboxyl terminus of the human immunodeficiency virus type I gp120 glycoprotein to CD4 binding.

The carboxyl half of the HIV-1 gp120 glycoprotein, which has been implicated in binding to the CD4 receptor, contains two disulfide bonds linking cysteine residues 378-445 and 385-418. To examine the necessity of these disulfide bonds for the formation and/or maintenance of a gp120 glycoprotein competent for CD4 binding, we created mutants of a soluble form of gp120 in which combinations of these cysteine residues were altered. The mutant glycoproteins were examined for export from the expressing cell and for CD4 binding ability. Mutant gp120 molecules lacking both disulfide bonds were not stably expressed or exported. However, mutants for which either disulfide bond could form were exported and were fully competent for CD4 binding. In some cases, the presence of one of the pair of linked cysteines exerted more detrimental effects on export or CD4 binding than did alteration of both cysteines. Thus, the evaluation or the contribution of a particular disulfide bond to a phenotype should include studies in which both cysteines involved in the bond are simultaneously altered.

CD4 Antigens↗
PubMed1990

Determination and metabolism of dithiol chelating agents: X. In humans, meso-2,3-dimercaptosuccinic acid is bound to plasma proteins via mixed disulfide formation.

meso-2,3-Dimercaptosuccinic acid (DMSA) is orally effective for the treatment of chronic lead intoxication in humans. Earlier studies have shown that the majority of DMSA, given p.o. to normal humans, is excreted in the urine as mixed disulfides with L-cysteine. We have developed an assay for the determination of DMSA that has made possible the determination of the form of DMSA in blood and plasma. After p.o. administration of 10 mg DMSA/kg to four normal young men, no unaltered DMSA (unaltered DMSA is the unbound, parent compound; total DMSA consists of unaltered DMSA plus oxidized (disulfide) DMSA and is determined after reduction with dithiothreitol) was found in the blood over an 8-hr period. Only after treatment of blood or plasma with the disulfide-reducing agent, dithiothreitol, was DMSA detected. This indicates that DMSA is in disulfide linkage with plasma proteins and/or non-protein sulfhydryl compounds. Most of the DMSA in the plasma (92-95%) was found to be bound to plasma proteins, mainly albumin. The remaining DMSA may be bound to small molecular weight (less than 10,000 MW) nonprotein sulfhydryl compounds such as cysteine. Plasma protein appears to serve as a depot and reservoir of DMSA, which can exchange for cysteine. The urinary excretion of unaltered DMSA and DMSA mixed disulfides with L-cysteine suggests that this exchange takes place at the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1986

The autotrophic pathway of acetogenic bacteria. Role of CO dehydrogenase disulfide reductase.

An enzyme from Clostridium thermoaceticum has been isolated which reduces disulfides of carbon monoxide dehydrogenase and it has been named CO dehydrogenase disulfide reductase. The enzyme is a tetramer of molecular weight 225,000 made up of four apparently identical monomers. It does not contain methionine or tryptophan and contains 2 calcium and 1 zinc/monomer. NADP or ferredoxin serves as an electron carrier. This enzyme is part of the system that permits certain bacteria to grow with CO or CO2 and H2 as the source of carbon and energy. The portion of the pathway which is being investigated is the conversion of methyltetrahydrofolate, CO, and CoASH to acetyl-CoA. All the enzymes required for this synthesis have now been purified. In combination with CO dehydrogenase, CO dehydrogenase disulfide reductase with NADP or ferredoxin catalyzes a reversible exchange of [3H]CoASH with acetyl-CoA. The disulfide reductase apparently is involved in the portion of the pathway in which CoASH is introduced into the acetyl-CoA. In addition, the reductase activates CO dehydrogenase in the overall synthesis of acetyl-CoA from methyltetrahydrofolate, CO, and CoASH by reducing about one disulfide group/monomer of the alpha 3 beta 3 CO dehydrogenase. The above exchange reaction in combination with the observation that [14C]acetate is formed from CO and the 14CH3-[Co]corrinoid enzyme in the absence of CoASH have permitted ordering of the sequence of reactions by which CO dehydrogenase plays a central role in the autotrophic synthesis of acetyl-CoA.

Acetyl Coenzyme A↗
PubMed1988

Complexing activity of 2,3-dimercapto-1-propanesulfonate and its disulfide auto-oxidation product in rat kidney.

The complexing activity of 2,3-dimercapto-1-propanesulfonate (DMPS) was examined in vivo and in the isolated perfused rat kidney. Intravenous infusions of the sulfhydryl form of DMPS into rats pretreated with HgCl2 resulted in a dose-dependent increase in the urinary excretion of mercury and a decrease in the retention of mercury in kidney. A 120-min infusion of 2 mg sulfhydryl DMPS.min-1.kg body wt-1 resulted in excretion of 76% of the mercury body burden of which greater than 90% was removed from kidney. The disulfide auto-oxidation product of DMPS significantly increased the urinary excretion of mercury in the rat, but its effect was less than that of an equivalent dose of sulfhydryl DMPS. The presence of disulfide DMPS or 3:1 mixtures of sulfhydryl DMPS and disulfide DMPS in the perfusate increased the excretion of mercury and induced a loss of mercury from the cortex and outer medulla in the isolated perfused rat kidney. Thus both the disulfide and sulfhydryl forms of DMPS can act directly on the kidney to accelerate the excretion of mercury. This is consistent with previous observations of the ability of the kidney to reduce disulfide DMPS to sulfhydryl DMPS.

Animals↗
PubMed1982

Coenzyme A thiosulfonate (coenzyme A disulfide-S,S-dioxide), an affinity analog of coenzyme A.

The structure of the CoA affinity analog-oxidized CoA disulfide (o-CoAS2) (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4939) has been deduced to be that of the thiosulfonate of CoA, i.e. coenzyme A disulfide-S,S-dioxide. This deduction is based on several considerations among which are: the cleavage of o-CoAS2 by dithiothreitol under anaerobic conditions to equimolar amounts of CoASH and CoASO2H; the alkali-catalyzed dismutation of 3 mol of o-CoAS2 to 4 mol of CoASO2H and 1 mol of CoA disulfide; and comparison of the 13C-NMR spectra of CoA disulfide and o-CoAS2. The results of studies with Clostridial phosphotransacetylase (EC 2.3.1.8) and pigeon muscle carnitine acetyltransferase (EC 2.3.1.7) were consistent with the action of o-CoAS2 as a CoA affinity analog on these enzymes. Inactivation was characterized by what appeared to be disulfide bonding between CoA and important sulfhydryl groups of the proteins.

Affinity Labels↗
PubMed1994

S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence against a role for glutathione disulfide.

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thio by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of the proteins did not occur by thiol/disulfide exchange. Approximately 10 mol% of one heavily modified band (29 kDa) was S-thiolated after 30 min. A second major band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolated after 30 min. These experiments identify a subset of S-thiolated neutrophil proteins, including actin, whose modification is related to the phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein-bound without an appreciable change in non-bound concentration of glutathione or glutathione disulfide. These results suggest that glutathione was synthesized during initial phases of the respiratory burst, compensating for the amount of glutathione that became protein-bound. Since there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation.

Actins↗
PubMed1998

Dithiocarbamate toxicity toward thymocytes involves their copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox cycle without the release of reactive oxygen species.

We have reported previously that diethyldithio-carbamate (DDC) and pyrrolidine dithiocarbamate (PDTC) induce apoptosis in rat thymocytes. Apoptosis was shown to be dependent upon the transport of external Cu ions into the cells and was accompanied by the oxidation of intracellular glutathione, indicating the inducement of pro-oxidative conditions (C. S. I. Nobel, M. Kimland, B. Lind, S. Orrenius, and A. F. G. Slater, J. Biol. Chem. 270, 26202-26208, 1995). In the present investigation we have examined the chemical reactions underlying these effects. Evidence is presented to suggest that dithiocarbamates undergo oxidation by CuII ions, resulting in formation of the corresponding thiuram disulfides, which are then reduced by glutathione, thereby generating the parent dithiocarbamate and oxidized glutathione (glutathione disulfide). Although DDC and PDTC were found to partially stabilize CuI ions, limited redox cycling of the metal ion was evident. Redox cycling did not, however, result in the release of reactive oxygen species, which are believed to be scavenged in situ by the dithiocarbamate. DDC and PDTC were, in fact, shown to prevent copper-dependent hydroxyl radical formation and DNA fragmentation in model reaction systems. The thiuram disulfide disulfiram (DSF) was found to induce glutathione oxidation, DNA fragmentation, and cell killing more potently than its parent dithiocarbamate, DDC. Of particular importance was the finding that, compared with DDC, the actions of DSF were less prone to inhibition by the removal of external copper ions with a chelating agent. This observation is consistent with our proposed mechanism of dithiocarbamate toxicity, which involves their copper-catalyzed conversion to cytotoxic thiuram disulfides.

Animals↗
PubMed1997

Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity.

We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

Feasibility Studies↗
PubMed1984

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins.

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.

Chemical Phenomena↗
PubMed1987

Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift.

An enzyme catalyzing thiol-disulfide exchange, thioltransferase, was purified to homogeneity from pig liver. By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.

Amino Acids↗
PubMed1993

The DsbA-DsbB system affects the formation of disulfide bonds in periplasmic but not in intramembraneous protein domains.

The DsbA and DsbB proteins of Escherichia coli are involved in facilitating the formation of disulfide bonds in periplasmic proteins. Here, we show that the rate of formation of a disulfide bond in the periplasmic domain of the inner membrane protein leader peptidase is reduced in dsbA and dsbB strains, whereas the rate of formation of a disulfide bond engineered into the membrane embedded domain of the same protein is completely unaffected by these mutations. We conclude that the Dsb proteins do not facilitate the formation of intramembraneous disulfides.

Bacterial Proteins↗
PubMed1993

The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity.

Protein disulfide isomerase (PDI) is believed to function in vivo by catalyzing the isomerization of disulfide bonds in proteins and thereby facilitating their folding. In S. cerevisiae PDI is encoded by an essential gene. Deletion of nearly one-third of the C-terminal residues of PDI altered PDI's cellular localization but not cell viability. Further deletions resulted in lethality, but these truncated proteins still exhibited PDI activity in vitro. Cells carrying a variant PDI in which both-CGHC-active sites were disrupted were viable. However, these cells exhibited a delay in the disulfide bond formation and transport of carboxypeptidase Y. In vitro enzyme assays revealed that disruption of both sites abolished catalytic activity. These results indicate that PDI catalyzes disulfide bond formation both in vivo and in vitro and that the integrity of the active sites is required for catalysis. However, this catalytic activity is not essential for yeast viability.

Amino Acid Sequence↗