Biological regulation through protein disulfide bond cleavage

It is thought that disulfide bonds in secreted proteins are inert because of the oxidizing nature of the extracellular milieu. We have suggested that this is not necessarily the case and that certain secreted proteins contain one or more disulfide bonds that can be cleaved and that this cleavage is central to the protein's function. This review discusses disulfide bond cleavage in the secreted soluble protein, plasmin. Cleavage of plasmin disulfide bond(s) triggers peptide bond cleavage and formation of the tumour angiogenesis inhibitor, angiostatin. Tumour cells secrete phosphoglycerate kinase which facilitates cleavage of the plasmin disulfide bond(s). Phosphoglycerate kinase is not a conventional disulfide bond reductase. We propose that phosphoglycerate kinase facilitates cleavage of a particular plasmin disulfide bond by hydroxide ion, which results in formation of a sulfenic acid and a free thiol. The free thiol is then available to exchange with another nearby disulfide bond resulting in formation of a new disulfide and a new free thiol. The reduced plasmin is then susceptible to discreet proteolysis which results in release of angiostatin.     

Characterization of a Reduced Form of Plasma Plasminogen as the Precursor for Angiostatin Formation

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys⁴⁶²-Cys⁵⁴¹ disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys⁵⁴¹ thiolate anion on the Cys⁵³⁶ sulfur atom of the Cys⁵¹²-Cys⁵³⁶ disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys⁵¹²-Cys⁵³⁶ allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys⁴⁶²-Cys⁵⁴¹ and Cys⁵¹²-Cys⁵³⁶ disulfides have −/+RHHook and −LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the −/+RHHook and −LHHook disulfides, along with the −RHStaple bond, are potential allosteric configurations.     

Secretion of phosphoglycerate kinase from tumour cells is controlled by oxygen-sensing hydroxylases

Solid tumour cells employ glycolytic enzymes including phosphoglycerate kinase (PGK) to make ATP when their supply of oxygen is limiting. PGK is also secreted by tumour cells and facilitates cleavage of disulfide bonds in plasmin, which triggers proteolytic release of the angiogenesis inhibitor, angiostatin. Although PGK production by tumour cells was enhanced by hypoxia, its secretion was inhibited. Inhibition of secretion correlated with decrease in angiostatin formation by the tumour cells. In contrast, hypoxia did not inhibit the secretion of the angiogenesis activator, vascular endothelial cell growth factor (VEGF). PGK secretion was reversed by normoxia and was under control of the oxygen-sensing protein hydroxylases, as inhibitors of this class of enzymes mimicked the effect of hypoxia on PGK secretion. Direct hydroxylation of PGK was not the mechanism by which the protein hydroxylases controlled its secretion. These findings show that production and secretion of PGK are regulated separately and indicate that oxygen and the protein hydroxylases can control not only gene expression but also protein secretion.     

Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.     

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

Plasmin reduction by phosphoglycerate kinase is a thiol-independent process.

Phosphoglycerate kinase (PGK) is secreted by tumor cells and facilitates reduction of disulfide bond(s) in plasmin (Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869-873). The angiogenesis inhibitor, angiostatin, is cleaved from the reduced plasmin by a combination of serine- and metalloproteinases. The chemistry of protein reductants is typically mediated by a pair of closely spaced Cys residues. There are seven Cys in human PGK, and mutation of all seven to Ala did not appreciably affect plasmin reductase activity, although some of the mutations perturbed the tertiary structure of the protein. Cys-379 and Cys-380 are close to the hinge that links the N- and C-terminal domains of PGK. Alkylation/oxidation of Cys-379 and -380 by four different thiol-reactive compounds reduced plasmin reductase activity to 7--35% of control. Binding of 3-phosphoglycerate and/or MgATP to the N- and C-terminal domains of PGK, respectively, triggers a hinge bending conformational change in the enzyme. Incubation of PGK with 3-phosphoglycerate and/or MgATP ablated plasmin reductase activity, with half-maximal inhibitory effects at approximately 1 mm concentration. In summary, reduction of plasmin by PGK is a thiol-independent process, although either alkylation/oxidation of the fast-reacting Cys near the hinge or hinge bending conformational change in PGK perturbs plasmin reduction by PGK, perhaps by obstructing the interaction of plasmin with PGK or perturbing conformational changes in PGK required for plasmin reduction.     

Stability and structure-forming properties of the two disulfide bonds of alpha-conotoxin GI

alpha-Conotoxin GI is a 13 residue snail toxin peptide cross-linked by Cys2-Cys7 and Cys3-Cys13 disulfide bridges. The formation of the two disulfide bonds by thiol/disulfide exchange with oxidized glutathione (GSSG) has been characterized. To characterize formation of the first disulfide bond in each of the two pathways by which the two disulfide bonds can form, two model peptides were synthesized in which Cys3 and Cys13 (Cono-1) or Cys2 and Cys7 (Cono-2) were replaced by alanines. Equilibrium constants were determined for formation of the single disulfide bonds of Cono-1 and Cono-2, and an overall equilibrium constant was measured for formation of the two disulfide bonds of alpha-conotoxin GI in pH 7.00 buffer and in pH 7. 00 buffer plus 8 M urea using concentrations obtained by HPLC analysis of equilibrium thiol/disulfide exchange reaction mixtures. The results indicate a modest amount of cooperativity in the formation of the second disulfide bond in both of the two-step pathways by which alpha-conotoxin GI folds into its native structure at pH 7.00. However, when considered in terms of the reactive thiolate species, the results indicate substantial cooperativity in formation of the second disulfide bond. The solution conformational and structural properties of Cono-1, Cono-2, and alpha-conotoxin GI were studied by 1H NMR to identify structural features which might facilitate formation of the disulfide bonds or are induced by formation of the disulfide bonds. The NMR data indicate that both Cono-1 and Cono-2 have some secondary structure in solution, including some of the same secondary structure as alpha-conotoxin GI, which facilitates formation of the second disulfide bond by thiol/disulfide exchange. However, both Cono-1 and Cono-2 are considerably less structured than alpha-conotoxin GI, which indicates that formation of the second disulfide bond to give the Cys2-Cys7, Cys3-Cys13 pairing induces considerable structure into the backbone of the peptide.     

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.     

Binding of secretory component to dimers of immunoglobulin A in vitro. Mechanism of the covalent bond formation.

A complex between secretory component and an immunoglobulin A (IgA) myeloma dimer has been studied in vitro as a model to elucidate the mechanism of the formation of disulfide bonds during assembly in vivo of secretory immunoglobin A. A small amount of free thiol groups, totally about 0.4 groups per mole of protein, were shown to be present on both the heavy and light chains of the IgA dimer, but not on its J-chain, while no such groups could be demonstrated on free secretory component. The SH-groups on IgA most likely exist as a result of incomplete oxidation of some intra-or interchain disulfide bonds of the molecule, analogous to what has been suggested for IgG. Several types of evidence indicated that the disulfide bonds between secretory component and IgA are formed after the noncovalent association of the two proteins by a sulfhydryl group-disulfide bond exchange reaction, in which the small amount of free sulfhydryl groups on the IgA dimer initiate the reaction by reducing a reactive disulfide bond on secretory component. This exchange reaction, which thus proceeds by the mechanism of so-called disulfide interchange reactions, requires certain conformational features of one or both of the proteins and leads to the formation of presumably two new interchain disulfide bonds between secretory component and IgA. The reaction does not progress to completion, however, but ends in an equilibrium so that a small proportion of the secretory component molecules always are unattached by disulfide bonds.     

Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N- and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra- and inter-chain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.     

Reduction of a disulfide bond of thrombospondin in the supernatant solution of activated platelets

Incubation of the material secreted by activated platelets leads to the formation of disulfide-linked dimers and multimers of one of the proteins, thrombospondin. To determine whether these complexes formed as a result of thiol-disulfide exchange (no change in the number of thiols) or of oxidation of thiols (a decrease in the number of thiols), the number of thiols in TSP was measured during formation of multimers. The number of thiols increased from about 3/mol to 4.8/mol. The half-time for the disappearance of monomers of thrombospondin was fourfold greater than the half-time for appearance of new thiols. The appearance of new thiols, as well as the formation of multimers, was inhibited by Ca2+. The appearance of new thiols was reversible; addition of Ca2+ reversed the process, and at pH 8, but not at pH 6 or 7, the appearance of new thiols spontaneously reversed. No new thiols formed during incubation of partially purified thrombospondin or after the supernatant solution had been treated with activated thiol-Sepharose to remove reactive thiol compounds. It is concluded that thrombospondin has a disulfide bond that is unstable in the absence of Ca2+. It can be attacked by a thiol of another molecule of thrombospondin to form disulfide-linked multimers, by a thiol of the same molecule of thrombospondin to generate isomerization of disulfide bonds or, as observed in this study, by another secreted thiol compound to give a mixed disulfide and a new thiol.     

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-α mutant by Pichia pastoris

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-α mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of β-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.     

Disulfide bonds as switches for protein function

The prevailing view is that disulfide bonds have been added during evolution to enhance the stability of proteins that function in a fluctuating cellular environment. However, recent evidence indicates that disulfide bonds can be more than inert structural motifs. The function of some secreted soluble proteins and cell-surface receptors is controlled by cleavage of one or more of their disulfide bonds; this cleavage is mediated by catalysts or facilitators that are specific for their substrate.     

Annexin A2-S100A10 heterotetramer, a novel substrate of thioredoxin

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.     

SNAP-25 Contains Non-Acylated Thiol Pairs that can Form Intrachain Disulfide Bonds: Possible Sites for Redox Modulation of Neurotransmission

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.     

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.     

Pyrroloquinoline quinone (coenzyme PQQ) and the oxidation of SH residues in proteins.

Pyrroloquinoline quinone (PQQ) catalyzes the oxidation of cysteamine at neutral pH with a second order rate constant K2 = 0.45 M-1 s-1. The reduction of PQQ was monitored by absorption and fluorescence spectroscopy, whereas the oxidation of cysteamine to cystamine was followed by titration with 5,5'-dithiobis(2-nitrobenzoic acid). PQQ also catalyzes the oxidation of thiol groups critically connected with the function of two proteins, i.e. thioredoxin and phosphoribulose kinase. The reaction of PQQ with reduced thioredoxin brings about the oxidation of two thiol groups of the oxireductase, whereas the enzyme phosphoribulose kinase is inactivated at 25 degrees C. The oxidized disulfide bond of phosphoribulose kinase is reduced by dithiothreitol and the enzyme recovers catalytic activity. The ability of PQQ to catalyze the oxidation of vicinal cysteinyl residues to generate disulfide bonds under mild experimental conditions can be exploited to define the precise role of modified thiol residues in either catalysis or stabilization of protein structure.