A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.     

Function of Glu-469' in the acid-base catalysis of thioredoxin reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Because dipteran insects such as Drosophila melanogaster lack glutathione reductase, their TrxRs are particularly important for antioxidant protection; reduced Trx reacts nonenzymatically with oxidized glutathione to maintain a high glutathione/glutathione disulfide ratio. Like other members of the pyridine nucleotide-disulfide oxidoreductase family, TrxR is a homodimer; in the enzyme from D. melanogaster (DmTrxR), each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57-Cys-62 redox-active disulfide from one monomer and a Cys-489'-Cys-490' C-terminal redox-active disulfide from the second monomer. A dyad of His-464' and Glu-469' in TrxR acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis [Huang, H.-H., et al. (2008) Biochemistry 47, 1721-1731]. In this investigation, the role of Glu-469' in catalysis by DmTrxR has been studied. The E469'A and E469'Q DmTrxR variants retain 28 and 35% of the wild-type activity, respectively, indicating that this glutamate residue is important but not critical to catalysis. The pH dependence of V(max) for both glutamate variants yields pK(a) values of 6.0 and 8.7, compared to those in the wild-type enzyme of 6.4 and 9.3, respectively, indicating that the basicity of His-464' in TrxR in complex with its substrate, DmTrx-2, is significantly lower in the glutamate variants than in wild-type enzyme. The rates of some steps in the reductive half-reactions in both glutamate variants are much slower than those of the wild-type enzyme. On the basis of our observations, it is proposed that the function of Glu-469' is to facilitate the positioning of His-464' toward the interchange thiol, Cys-57, as suggested for the analogous residue in glutathione reductase.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae.     

Activation of translation via reduction by thioredoxin-thioredoxin reductase in Saccharomyces cerevisiae

Previously we reported that in vitro translation activity in extracts of Saccharomyces cerevisiae was stimulated by dithiothreitol (DTT) and further increased by the addition of thioredoxin (TRX1) [Choi, S.K. (2007) Thioredoxin-mediated regulation of protein synthesis by redox in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 35, 36-40]. To identify the pathway affecting translation, we cloned and purified thioredoxin reductase 1 (TRR1), thioredoxin reductase 2 (TRR2), glutaredoxin 1 (GRX1) and glutaredoxin reductase 1 (GLR1) as fusion proteins. Thioredoxin-mediated activation of translation was more effectively stimulated by NADPH or NADH than by DTT. Moreover, addition of TRR1 led to a further increase of translation in the presence of thioredoxin plus NADPH. These findings indicate that redox control via the thioredoxin-thioredoxin reductase system plays an important role in the regulation of translation.     

Acid-base catalysis in the mechanism of thioredoxin reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide oxidoreductase enzyme family, the enzyme from Drosophila melanogaster is a homodimer, and each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer and a Cys-489'/Cys-490' C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, thioredoxin reductase (DmTrxR) is particularly important; in addition to its normal functions, it also reduces GSSG for antioxidant protection. DmTrxR, used as a model for the enzyme from the malaria vector, Anopheles gambiae, has been shown to cycle in catalysis between the two-electron and four-electron reduced states, EH2 and EH4 [Bauer, H. et al. (2003) J. Biol. Chem. 278, 33020-33028]. His-464' acts as an acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis. The H464'Q enzyme has only 2% of the wild-type activity, emphasizing the importance of this residue. The pH dependence of Vmax for wild-type DmTrxR has pKa values of 6.4 and 9.3 on the DmTrxR-DmTrx-2 complex, whereas H464'Q DmTrxR only has an observable pKa at 6.4, indicating that the pKa at pH 9.3 is contributed mainly by His-464'. The pKa at pH 6.4 has been assigned to Cys-57 and Cys-490'; the thiolate on Cys-490' is the nucleophile in the reduction of Trx. In contrast to wild-type DmTrxR, H464'Q DmTrxR does not stabilize a thiolate-FAD charge-transfer complex in the presence of excess NADPH. The rates of steps in both the reductive and the oxidative half-reactions are markedly diminished in H464'Q DmTrxR as compared to those of wild-type enzyme, indicating that His-464' is involved in both half-reactions.     

Reduction of purothionin by the wheat seed thioredoxin system

Thioredoxin h, the thioredoxin characteristic of heterotrophic plant tissues, was purified to homogeneity from wheat endosperm (flour) and found to resemble its counterpart from carrot cell cultures. In the presence of NADPH, homogeneous thioredoxin h and partially purified wheat endosperm thioredoxin reductase (NADPH), (EC 1.6.4.5), purothionin promoted the activation of chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). Under these conditions, NADPH provided the reducing equivalents for a series of thiol reactions in which (a) thioredoxin reductase reduced thioredoxin h thereby converting it from disulfide (S-S) to sulfhydryl (SH) form; (b) the sulfhydryl form of thioredoxin h reduced the disulfide form of purothionin—a 5 kilodalton seed storage protein with 4 S-S bridges; and (c) the sulfhydryl form of purothionin reductively activated fructose-1,6-bisphosphatase. The results show that, since thioredoxin h does not react effectively with fructose-1,6-bisphosphatase, the thioredoxin system can activate an enzyme through purothionin by secondary thiol redox control. In a related type reaction, purothionin, inhibited the activity of either Escherichia coli or calf thymus ribonucleotide reductase with reduced thioredoxin as hydrogen donor. The results suggest that purothionin competes with ribonucleotide reductase for reducing equivalents from thioredoxin. Thus, inhibition of deoxyribonucleotide synthesis should be considered a possible mechanism when examining the toxic effects of purothionin on mammalian cells in S-phase.     

Thioredoxin 1 Is Inactivated Due to Oxidation Induced by Peroxiredoxin under Oxidative Stress and Reactivated by the Glutaredoxin System

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H₂O₂. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.     

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.     

Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen.

The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox-active dithiols. We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 microM selenite plus 0.05 microM calf thymus thioredoxin reductase at pH 7.5 caused a non-stoichiometric oxidation of NADPH (100 microM after 30 min). In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 microM E. coli thioredoxin also resulted in non-stoichiometric oxidation of NADPH, consistent with oxidation of the two active-site thiol groups in thioredoxin to a disulfide. Kinetically, the reaction was complex with a lag phase at low selenite concentrations. Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s). Ferricytochrome c was effectively reduced by calf thymus thioredoxin reductase and selenite in the presence of oxygen. Selenite caused a strong dose-dependent inhibition of the formation of thiol groups from insulin disulfides with either the E. coli or calf-thymus thioredoxin system. Thus, under aerobic conditions selenite catalyzed, NADPH-dependent redox cycling with oxygen, a large oxygen-dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide-reductase activity.     

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.     

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.     

Enzymes of thiol-disulfide metabolism of proteins

Occurrence, properties and physiological role of protein disulfide reductases (EC 1.6.4.4 and 1.8.4.2), protein disulfide isomerase (EC 5.3.4.1), and thiol oxidase (EC 1.8.3.2) catalyzing thiol-disulfide interchange reactions in proteins are reviewed with a particular emphasis on seed storage proteins. An important role of the enzymes in the formation and degradation of seed storage protein complexes is discussed.     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.     

Titration studies on the active sites of pig heart lipoamide dehydrogenase and yeast glutathione reductase as monitored by the charge transfer absorbance

Macroscopic pKa values associated with the influence of pH on the visible spectrum of 2-electron reduced pig heart lipoamide dehydrogenase and yeast glutathione reductase have been determined by monitoring changes in the principal flavin band near 460 nm and the charge transfer band at 540 nm. The ionization of at least three active site amino acid side chains can influence the spectra over the range of pH studied: the two nascent thiols (interchange thiol and electron transfer thiol) and the histidine residue which acts as the base catalyst in lipoamide dehydrogenase and the acid catalyst in glutathione reductase thiol-disulfide interchange reactions. These systems are analogous to, but more complex than, those in glyceraldehyde-3-phosphate dehydrogenase and papain where a single thiol and a histidine residue in a relatively apolar milieu form a thiolate-imidazolium ion pair which is favored over the thiol-imidazole prototropic tautomer. In an effort to more nearly mimic the papain titrations, the macroscopic pKa values were determined on reduced glutathione reductase which had been monoalkylated with iodoacetamide under conditions known to favor the reaction of the interchange thiol by at least 10 to 1 (Arscott, L. D., Thorpe, C., and Williams, C. H., Jr. (1981) Biochemistry 20, 1513-1520). Like papain and glyceraldehyde-3-phosphate dehydrogenase, alkylated glutathione reductase showed two macroscopic pKa values, at pH 3.7 and pH 9.1, and by analogy, these were associated primarily with the thiol and the imidazole, respectively. Results with the native enzymes depended on the wavelength monitored. Glutathione reductase had pKa values at 4.8, 7.1, and 9.2 when monitored at 540 nm and 5.1 and 8.2 when monitored at 462 nm. Lipoamide dehydrogenase had pKa values at 4.4 and 8.7 when monitored at 529 nm and 3.9, 7.0, and 9.3 when monitored at 455 nm.