EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes

Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts

The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.     

Calcium regulates thioredoxin reductase in human metastatic melanoma

Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.     

Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes.

Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin. The modified thioredoxins alone are inactive. These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.     

Reactions of anthranilate hydroxylase with salicylate, a nonhydroxylated substrate analogue. Steady state and rapid reaction kinetics

Steady state and rapid reaction kinetics of the flavoprotein anthranilate hydroxylase (EC 1.14.12.2) have been examined with the nonhydroxylated substrate analogue, salicylate. Since the reaction with salicylate does not involve events in which aromatic substrate is oxygenated, it provides a simpler model for studying the hysteresis exhibited by this enzyme. It is shown that the first turnover of the enzyme is slower than subsequent turnovers owing in part to slow initial binding reactions of salicylate with the enzyme. The reductive half-reaction of the first turnover is also slow since rapid reduction of the enzyme flavin requires bound aromatic substrate. The oxidative half-reaction involves reaction of the reduced enzyme-salicylate complex with oxygen to form a flavin C4a-hydroperoxide, which then decays to oxidized flavoenzyme and H2O2. Several lines of evidence indicate that salicylate remains bound to the enzyme at the end of the catalytic cycle so that in turnovers subsequent to the first, the slow steps involving salicylate binding are avoided.     

The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange

The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein.     

The role of thioredoxin reductase in the reduction of free radicals at the surface of the epidermis

A study of guinea pig and human skin in vivo has revealed that keratinocytes contain a thioenzyme which reduces radicals. This enzyme has been purified by affinity column chromatography and identified as thioredoxin reductase. In vivo and in vitro bioassays were performed by using a spin-labelled surfactant as the radical substrate, because it can diffuse through the stratum corneum and react by surface complexation with the epidermis and also on the outer plasma membrane of keratinocytes from cell cultures. Thioredoxin, the native substrate for thioredoxin reductase effectively competes for electrons with radical substrates. Nicotinamide adenine dinucleotide phosphate (NADPH) is the electron donating coenzyme in both the reduction of radicals and thioredoxin. Reduced thioredoxin has been shown to be an inhibitor of tyrosinase, whereas oxidized thioredoxin has no effect on this enzyme activity. Taken together these results indicate that the thioredoxin/thioredoxin reductase system plays an important role in preventing cell damage from UV-generated free radicals on the skin.     

How thioredoxin can reduce a buried disulphide bond

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.     

Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP

Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups. The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity. The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.     

The kinetics of unphosphorylated, phosphorylated and proteolytically modified fructose bisphosphatase from fat liver

Phosphorylation of fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) by the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle decreased the K0.5 for fructose-bisphosphate from 21 to 11 microM. When the phosphorylated fructose-bisphosphatase was treated with trypsin the K0.5 increased to 22 microM. The K0.5 also increased when the phosphoenzyme was treated with a partially purified phosphatase from rat liver. There was no difference between the unphosphorylated and phosphorylated enzyme with respect to pH dependence, the pH optimum being about 7.0 for both. Limited treatment of fructose-bis-phosphatase with subtilisin, which cleaves the enzyme at its unphosphorylatable N-terminal part, increased the pH optimum more than limited treatment with trypsin, which releases the phosphorylated peptide at the C-terminal part of fructose-bisphosphatase. The phosphorylated site on the phosphorylated fructose-bisphosphatase was more easily split off by trypsin treatment than the corresponding unphosphorylated site. The results suggest in addition to the glucagon-induced phosphorylation of fructose-bisphosphatase described by Claus et al. [1] that the phosphorylation-dephosphorylation of fructose-bisphosphatase could be of importance for the hormonal regulation of the enzyme in vivo.     

Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.     

The enzymology and biochemistry of methionine sulfoxide reductases

The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.     

Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.     

Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) 相似文献   

Time-dependent irreversible inhibition of bovine kidney alkaline phosphatase by oxidized adenosine. Use of this compound as a site-directed inhibitor for studying uncompetitive inhibition

The L/B/K type of mammalian alkaline phosphatase (ALP) is inhibited uncompetitively by nucleotides. A combination of adenosine and nicotinamide is more effective than either adenosine or nicotinamide alone, probably because a dinucleotide structure is necessary to trigger a conformational change accompanying binding of structures such as NADH. It has been suggested that a loop region containing residue 429 in the ALP polypeptide is important in the interaction of uncompetitive inhibitors with the enzyme. In the L/B/K isoenzyme, residue 429 is a histidine and is a potential target for modification. In an attempt to learn more about the molecular events accompanying inhibition of ALP by uncompetitive inhibitors, bovine kidney ALP was reacted with oxidized adenosine in the presence of nicotinamide to see if site-directed modification occurs. Kidney ALP was irreversibly inactivated by oxidized adenosine but the reaction was slow. The site modified is likely to be close to the region of binding. Sequence data for the kidney enzyme shows that in the region of residue 429 there are no residues except His⁴²⁹ itself that is likely to react with oxidized adenosine.     

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.     

Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase.

Selenium compounds like selenite (SeO3(2-) may form a covalent adduct with glutathione (GSH) in the form of selenodiglutathione (GS-Se-SG), which is assumed to be important in the metabolism of selenium. We have isolated GS-Se-SG and studied its reactions with NADPH and thioredoxin reductase from calf thymus or with thioredoxin reductase and thioredoxin from Escherichia coli. Incubation of 0.1 microM calf thymus thioredoxin reductase or 0.1 microM thioredoxin reductase and 1 microM thioredoxin from E. coli with 5, 10, or 20 microM GS-Se-SG resulted in a fast initial reaction, followed by a large and continued oxidation of NADPH. However, anaerobic incubation of 0.1 microM calf thymus thioredoxin reductase and 20 microM GS-Se-SG resulted only in oxidation of a stoichiometric amount of NADPH; admission of oxygen started continuous NADPH oxidation. Contrary to the mammalian enzyme, GS-Se-SG was not a substrate for thioredoxin reductase from E. coli. The rate of the oxygen-dependent reaction between calf thymus thioredoxin reductase and GS-Se-SG was increased 2-fold in the presence of 4 mM GSH, indicating that HSe- was the reactive intermediate. Glutathione reductase from rat liver reduced GS-Se-SG with a very slow continued oxidation of NADPH, and the presence of the enzyme did not affect the oxygen-dependent nonstoichiometric oxidation of NADPH by GS-Se-SG and thioredoxin reductase. Fluorescence spectroscopy showed GS-Se-SG to be a very efficient oxidant of reduced thioredoxin from E. coli and kinetically superior to insulin disulfides. Thioredoxin-dependent reduction of CDP to dCDP by ribonucleotide reductase was effectively inhibited by GS-Se-SG.     

Enzyme regulation in C4 photosynthesis: Mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using ¹⁴C-iodoacetate and ¹⁴C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.