Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.     

Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the beta-oxidation pathway.

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.     

Steady-state kinetics of 3-mercaptopyruvate sulfurtransferase from bovine kidney

Mammalian 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), purified to apparent homogeneity by a new procedure, was studied by steady-state kinetic methods. The enzyme-catalyzed transfer of a sulfur atom from 3-mercaptopyruvate either to 2-mercaptoethanol or to a second molecule of 3-mercaptopyruvate was found to proceed by a sequential formal mechanism. An overall mechanism incorporating both of these transfers was shown to be capable of generating all of the initial velocity and product inhibition behavior observed.     

Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria.

In rat liver hypo-osmotically treated mitochondria, 2-mercaptoacetate inhibits respiration induced by palmitoyl-CoA, octanoate or butyryl-CoA only when the reaction medium is supplemented with ATP. Under this condition, NADH-stimulated respiration is not affected. In liver mitochondrial matrix, the presence of ATP is also required to observe a 2-mercaptoacetate-induced inhibition of acyl-CoA dehydrogenases tested with palmitoyl-CoA, butyryl-CoA or isovaleryl-CoA as substrate. As the oxidation of these substrates is also inhibited by the incubation medium resulting from the reaction of 2-mercaptoacetate with acetyl-CoA synthase, with conditions under which 2-mercaptoacetate has no effect, 2-mercaptoacetyl-CoA seems to be the likely inhibitory metabolite responsible for the effects of 2-mercaptoacetate. Kinetic experiments show that the main effect of the 2-mercaptoacetate-active metabolite is to decrease the affinities of fatty acyl-CoA dehydrogenases towards palmitoyl-CoA or butyryl-CoA and of isovaleryl-CoA dehydrogenase towards isovaleryl-CoA. Addition of N-ethylmaleimide to mitochondrial matrix pre-exposed to 2-mercaptoacetate results in the immediate reversion of the inhibitions of palmitoyl-CoA and isovaleryl-CoA dehydrogenations and in a delayed reversion of butyryl-CoA dehydrogenation. These results led us to conclude that (i) the ATP-dependent conversion of 2-mercaptoacetate into an inhibitory metabolite takes place in the liver mitochondrial matrix and (ii) the three fatty acyl-CoA dehydrogenases and isovaleryl-CoA dehydrogenase are mainly competitively inhibited by this compound. Finally, the present study also suggests that the inhibitory metabolite of 2-mercaptoacetate may bind non-specifically to, or induce conformational changes at, the acyl-CoA binding sites of these dehydrogenases.     

The crystal structure of Leishmania major 3-mercaptopyruvate sulfurtransferase. A three-domain architecture with a serine protease-like triad at the active site

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions.     

Metabolism ofl-cysteine via transamination pathway (3-mercaptopyruvate pathway)

Summary We have studied the transamination pathway (3-mercaptopyruvate pathway) ofl-cysteine metabolism in rats. Characterization of cysteine aminotransferase (EC 2.6.1.3) from liver indicated that the transamination, the first reaction of this pathway, was catalyzed by aspartate aminotransferase (EC 2.6.1.1). 3-Mercaptopyruvate, the product of the transamination, may be metabolized through two routes. The initial reactions of these routes are reduction and transsulfuration, and the final metabolites are 3-mercaptolactate-cysteine mixed disulfide [S-(2-hydroxy-2-carboxyethylthio)cysteine, HCETC] and inorganic sulfate, respectively. The study using anti-lactate dehydrogenase antiserum proved that the enzyme catalyzing the reduction of 3-mercaptopyruvate was lactate dehydrogenase (EC 1.1.1.27). Formation of HCETC was shown to depend on low 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) activity. Results were discussed in relation to HCETC excretion in normal human subjects and patients with 3-mercaptolactate-cysteine disulfiduria. Incubation of liver mitochondria withl-cysteine, 2-oxoglutarate and glutathione resulted in the formation of sulfate and thiosulfate, indicating that thiosulfate was formed by transsulfuration of 3-mercaptopyruvate and finally metabolized to sulfate.     

Biosynthesis of S-(2-hydroxy-2-carboxyethylthio)-L-cysteine (3-mercaptolactate-cysteine disulfide) by the rat heart

Incubation of 3-mercaptopyruvate with rat heart homogenate resulted in the formation of S-(2-hydroxy-2-carboxy-ethylthio)-L-cysteine (HCETC, 3-mercaptolactate-cysteine disulfide), L-cysteine and 3-mercaptolactate with the concomitant decrease in glutamate and aspartate. These results indicate that a part of 3-mercaptopyruvate was converted to L-cysteine by transamination, a part was reduced to 3-mercaptolactate, and HCETC was formed from these two products. Another peak which corresponds to L-cysteine-glutathione disulfide on amino acid analysis was also produced during the incubation.     

Affinity labeling of a catalytic site, cysteine(247), in rat mercaptopyruvate sulfurtransferase by chloropyruvate as an analog of a substrate

A bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (EC 2.8.1.2), is inactivated by 3-chloropyruvate, an analog of 3-mercaptopyruvate serving as a sulfur-donor and -acceptor substrate. To elucidate a reaction mechanism of the enzyme, the inactivation kinetic studies using 3-chloropyruvate were carried out. However, 3-chloropyruvate cannot be mixed with 3-mercaptopyruvate, 2-mercaptoethanol and thiosulfate because these substrates decompose 3-chloropyruvate. Thus, 3-mercaptopyruvate sulfurtransferase was incubated with 3-chloropyruvate, and then the remaining activity was measured separately in the assay system containing 3-mercaptopyruvate and 2-mercaptoethanol. The inactivation kinetics was analyzed by Kitz and Wilson method (J. Biol. Chem. 237 (1962) 3245-3248). The inactivation of mercaptopyruvate sulfurtransferase by 3-chloropyruvate proceeded in one-on-one manner and exhibited pseudo first-order kinetics with k(inact) = 0.068 +/- 0.003 min(-1) and K(I) = 4.0 +/- 0.2 mM (n = 3, mean +/- S.D.). Further, SH titration using DTNB revealed that MST was inactivated by 3-chloropyruvate in a 1:1 stoichiometry. Site-directed mutagenesis for binding sites of 3-mercaptopyruvate (Arg(187)-->Gly or Arg(196)-->Gly) (J. Biol. Chem. 271 (1996) 27395-27401) did not critically affect the inactivation. These findings suggest that 3-chloropyruvate behaves as an affinity label and directly tags the catalytic site, Cys(247). An ESI-LC/Q-TOF mass spectrometric study suggests that a pyruvate adduct is formed at Cys(247), which mimics a reaction intermediate.     

Desulfuration of l-cysteine through transamination and transsulfuration in rat liver

Desulfuration of L-cysteine by rat liver via a pathway of transamination followed by transsulfuration was studied using cyanide as a sulfur acceptor. More than a 5-fold increase in formation of thiocyanate from L-cysteine and cyanide was observed in the presence of 2-oxoglutarate and pyruvate. L-Cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase activities in the same preparations were also determined. It was concluded that L-cysteine was desulfurated through transamination and transsulfuration of the resulting 3-mercaptopyruvate, and that the rate-limiting step appears to be the transamination reaction.     

Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana.

Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule. Recently a sulfurtransferase specific for the substrate 3-mercaptopyruvate was isolated from Arabidopsis thaliana [Papenbrock, J. & Schmidt, A. (2000) Eur. J. Biochem. 267, 145-154]. In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously. Sequences of the mature proteins had an identity of 77.7%. The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases. When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases. The new sulfurtransferase was expressed in Escherichia coli fused with an N-terminal His6-tag, purified and tested for enzyme activity. Like the first enzyme, the newly isolated protein preferred 3-mercaptopyruvate to thiosulfate as substrate. The Km of both enzymes determined for 3-mercaptopyruvate and cyanide were almost identical. As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence- and stress-induced proteins. To prove the involvement of sulfurtransferases in senescence-associated processes 3-mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages. 3-mercaptopyruvate sulfurtransferase activity and steady-state RNA levels of sulfurtransferases increased with increasing age. However, steady-state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased. Putative roles of sulfurtransferases in senescence-associated processes are discussed.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria

Summary We have studied the 3-mercaptopyruvate pathway (transamination pathway) ofl-cysteine metabolism in rat liver mitochondria.l-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. Whenl-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withl-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation froml-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatl-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.     

Leishmania amazonensis: metabolic adaptations induced by resistance to an ABC transporter blocker

We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.     

Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat.

The contribution of cystathionine gamma-lyase, cystathionine beta-synthase and cysteine aminotransferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydration in rat liver and kidney was assessed with four different assay systems. Cystathionine gamma-lyase and cystathionine beta-synthase were active when homogenates were incubated with 280 mM-L-cysteine and 3 mM-pyridoxal 5'-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160 mM-L-cysteine, 2 mM-pyridoxal 5'-phosphate, 3 mM-2-oxoglutarate and 3 mM-dithiothreitol. At more-physiological concentrations of cysteine (2 mM) cystathionine gamma-lyase and cystathionine beta-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine gamma-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO4(2-) or [35S]taurine formed from labelled dietary cysteine.     

The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

H2S, a new neuromodulator

The formation of H2S from cyst(e)ine is catalyzed by three enzymes, cystathionine beta synthase, cystathionase, and 3-mercaptopyruvate sulfurtransferase. In the liver, kidney, enterocytes and vascular smooth muscle cells, H2S is principally synthesized by cystathionase. In contrast, it is synthesized by cystathionine beta synthase in the brain and partially by 3-mercaptopyruvate sulfurtransferase in cardiac tissue. H2S is catabolized, essentially in mitochondria by thiosulfate reductase. The sulfite generated is then oxidized to sulfate by sulfite oxidase. The amount of thiosulfate excreted in the urine is the best indicator of H2S biosynthesis, together with sulfhemoglobin determination in erythrocytes. H2S acts as a neuromodulator in the brain, increasing responses mediated by NMDA receptors, facilitating the induction of long-term potentialization in the hippocampus. H2S also acts as a vasodilator, acting directly on ATP-dependent potassium channels in vascular smooth muscle cells. The concentration of H2S is abnormally low in the brains of subjects with Alzheimer's disease, due to changes in the concentration of the physiological activator of cystathionine beta synthase. The overproduction of H2S described in subjects with Down's syndrome probably results from the overproduction of cystathionine beta synthase, as the gene encoding this protein is located on chromosome 21.     

Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.