Post-translational Regulation of Mercaptopyruvate Sulfurtransferase via a Low Redox Potential Cysteine-sulfenate in the Maintenance of Redox Homeostasis

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S 4 O 62 (cid:1) ) inhibited rat MST ( k i (cid:2) 3.3 min (cid:1) 1 , K i (cid:2) 120.5 (cid:3) M and k i (cid:2) 2.5min (cid:1) 1 , K i (cid:2) 178.6 (cid:3) M ,respectively).Theactivitywascompletely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys 247 , suggesting that Cys 247 is a target of the oxidants. A matrix-assisted laser desorption/ionization–time-of-flight mass spectro-metricanalysisrevealedthathydrogenperoxide-andtetrathionate- inhibited MSTs were increased in molecular mass consistent with theadditionofatomicoxygenandwithathiosulfate(S 2

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S 4 O 6 2؊ ) inhibited rat MST (k i ‫؍‬ 3.3 min ؊1 , K i ‫؍‬ 120.5 M and k i ‫؍‬ 2.5 min ؊1 , K i ‫؍‬ 178.6 M, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys 247 , suggesting that Cys 247 is a target of the oxidants. A matrixassisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide-and tetrathionateinhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S 2 O 3 ؊ ), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO ؊ ) at Cys 247 , which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.
We recently determined that MST is a housekeeping enzyme (36), and hydrogen peroxide did not change the amount of MST mRNA in Hep3B cells, 3 suggesting that MST activity is regulated at the enzymatic level. Mosharov et al. (37) have reported that in the cysteine anabolic pathway hydrogen peroxide promotes the activity of cystathionine ␤-synthase and suppresses the activity of methionine synthase at the transcriptional level, resulting in a facilitation of the metabolic flow to cystathionine. Consequently the amount of cysteine increased, and cellular reductants such as glutathione and thioredoxin were overproduced (38).
In this study, we provide evidence that MST is rapidly regulated at the enzyme level by the redox state, which in turn is associated with the control of cysteine degradation. These results suggest that MST helps maintain cellular redox homeostasis.

Preparation of Wild Type and Mutant
MSTs-Rat wild type and C247S (in which the catalytic site Cys 247 is replaced with serine) MST cDNAs were prepared according to a procedure described previously (11). C247S was used only in SH group titration. Complementary DNAs coding single mutant MSTs (C64S, C154S, C254S, and C263S) were synthesized by PCR using TaKaRa LA Taq with GC buffer (TAKARA BIO Inc. Otsu, Japan); wild type MST cDNA inserted in pBluescript (SKϩ) vectors (Stratagene, La Jolla, CA) was used as templates for mutant MSTs.
The PCR product was treated with DpnI and introduced into Escherichia coli XL1-Blue according to the usual protocol except for a preculture in LB medium without antibiotics at 37°C for 1 h before plating. Sequencing was performed to select each mutagenized cDNA using the synthesized antisense primers GCAGGTTCTGGCTCAGCCA, TGT-CCAGGTTCGATGCCATC, and AGGATGGTTCGGTGTCAC to examine the replacement of each of Cys 64 , Cys 154 , Cys 254 , and Cys 263 with serine, respectively. Each mutagenized cDNA of MST was digested from each construct (between the NcoI and XhoI sites) and was inserted into a pET28a vector (Novagen, San Diego, CA) (between the NcoI and XhoI sites).
Overexpression and Purification-Each pET28a vector containing cDNA of the wild type or mutant MST was introduced into E. coli, BL21 (DE3) cells transformed with a pSTV vector containing GroEL and GroES cDNAs (kindly provided by Dr. Matsumura, Department of Biochemistry and Molecular Biology, Nippon Medical School). The cells were cultured at 27°C in LB medium containing 30 g/ml kanamycin (Wako Pure Chemicals, Osaka, Japan). Isopropyl 1-thio-␤-D-galactopyranoside (Wako Pure Chemicals) was added at a final concentration of 1 mM when the A 600 reached 0.9. Cells were cultured at 37°C for 2.5 h and collected. Each recombinant MST was purified as described previously (21).
Inhibition and Inactivation of MSTs-Tetrathionate cannot be mixed with cyanide because the addition of cyanide to tetrathionate causes its decomposition. Thus, MST was first incubated with tetrathionate or hydrogen peroxide, and a 5-l aliquot was taken from the preincubation mixture. Then the remaining activity was measured separately in an assay system described under "Assay for Rhodanese Activity of MST." For studying the inhibition or inactivation of 20 M wild type MST, 20 M or 0.5 mM hydrogen peroxide (Wako Pure Chemicals) or tetrathionate (Nakalai Tesque, Kyoto, Japan) was incubated in 60 l of 20 mM potassium phosphate buffer, pH 7.4, on ice for a predetermined period of time (as described in each experiment). For studying the inhibition or inactivation of mutant enzymes, 20 M C64S, C154S, C254S, or C263S was incubated with 20 M or 0.5 mM hydrogen peroxide or tetrathionate in 60 l of 20 mM potassium phosphate buffer, pH 7.4, on ice for 1, 10, and 20 min.
The remaining activity is shown as a percentage of the activity of the untreated control enzyme.
Inhibition Kinetic Study-20 M MST and predetermined concentrations of hydrogen peroxide or tetrathionate (see figure legends) were incubated in 60 l of 20 mM potassium phosphate buffer, pH 7.4, on ice for a predetermined period of time (see figure legends). Then, a 5-l aliquot was taken from the preincubation mixture, and each rhodanese activity of MST was assayed.
The kinetic analysis was performed basically according to the Kitz and Wilson method (39). Each t1 ⁄ 2 value (time when the remaining activity is 50% of the untreated control activity) was determined from a semilog plot of v/v 0 versus time (v 0 being the reaction rate when inhibitor ϭ 0 mM). The k i value (inhibition rate constant) was determined from a replot of the t1 ⁄ 2 value versus 1/[I] (inhibitor).
This relationship can be represented by the following equations, where K i ϭ the dissociation constant of the EI complex, E ϭ enzyme, and EI ϭ enzyme-inhibitor noncovalent complex and The inhibition velocity equation is as follows.
Equations 2 and 3 can be rearranged to the following.
Integration of Equation 4 leads to the following, where E 0 ϭ the initial concentration of the enzyme. The ratio of the remaining enzyme activity can be represented as follows.
can be rearranged to the following.
Reactivation of Hydrogen Peroxide-and Tetrathionate-inhibited MSTs by DTT-After incubation of 20 M wild type and mutant MSTs with 20 M or 0.5 mM hydrogen peroxide, or 20 M or 0.5 mM tetrathionate in 60 l of 20 mM potassium phosphate buffer, pH 7.4, on ice for 20 min, free oxidants were removed from each sample and a control with a NAP5 column (Amersham Biosciences), and each mixture was concentrated to 20 M with a VIVASPIN (10,000MWCO. PES, Sartorius, Goettingen, Germany). A 5-l aliquot was taken from the mixture and mixed with 1 l of 10 mM (1.6 mM at a final concentration) DTT (Fluka, Osaka, Japan) solution. Each mixture was incubated at 25°C for 20 min.
Comparative Study for Reactivation of MST by DTT, Cysteine, Thioredoxin, or Glutathione-After incubation of 20 M wild type MST with 20 M tetrathionate or hydrogen peroxide on ice for 20 min in 60 l of 20 mM potassium phosphate buffer, pH 7.4, free oxidants were removed from each sample and a control with a NAP5 column. The enzyme-containing fractions were collected and concentrated to 20 M with a VIVASPIN.
In the experiment using L-cysteine (Kanto Kagaku, Tokyo, Japan) or DTT, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, or 102.4 mM cysteine or 1.6 mM DTT was added to 30 l of the mixture taken from the concentrated sample, and the total volume was adjusted to 60 l in 20 mM potassium phosphate buffer, pH 7.4. The mixture was incubated on ice for 20 min. After gel filtration with a NAP5 column, the enzyme-containing fractions were collected and concentrated to 20 M with a VIVASPIN. Then a 5-ml aliquot taken from the mixture was used for the assay of rhodanese activity In the experiment using recombinant E. coli thioredoxin (kindly provided by Dr. Abe, Department of Biochemistry and Molecular Biology, Nippon Medical School) or yeast glutathione (Sigma) with the reducing system, 30 l of the mixture taken from the concentrated MST sample was added to the reducing system. The reducing system contained 50 M NADPH (Sigma), 10 M thioredoxin or glutathione, and 0.2 M recombinant E. coli thioredoxin reductase (Dr. Abe) or 0.2 M glutathione reductase (Roche Applied Science) in 20 mM potassium phosphate buffer, pH 7.4. The total volume was adjusted to 60 l, and the mixture was incubated on ice for 20 min. After gel filtration, each activity was assayed in the same way as described above.
In the experiment using reduced thioredoxin or reduced glutathione, 2 l of 50 mM DTT was added to 198 l of 50 M thioredoxin and glutathione, and the mixture was incubated on ice for 20 min. After gel filtration with a NAP5 column, each sample was concentrated to 30 M with a Microcon-3 (Millipore). 30 l of the concentrated MST sample was mixed with 10 M reduced thioredoxin or reduced glutathione in 60 l of 20 mM potassium phosphate buffer, pH 7.4, and each mixture was incubated on ice for 20 min. After gel filtration, each activity was assayed in the same way as described above.
SH Group Titration-In the titration of the oxidized enzymes, after incubation of the wild type and five mutant MSTs (300 M) with a 300 M or 1.5 mM hydrogen peroxide in 30 l of 33 mM potassium phosphate buffer, pH 8.0, on ice for 20 min, free oxidants were removed from each sample with a NAP5 column.
In the titration of the reduced enzymes, oxygen in the solvent was removed using a glass apparatus by sequential evacuation and re-equilibration with oxygen-free argon. Oxygen-free argon was prepared by passing commercially obtained pure argon through a column of a Chromtopack Gas-Clean Oxygen Filter CP17970 (Varian, Inc., Palo Alto, CA). After incubation of 300 M wild type and the five mutant MSTs with 5 mM DTT in 30 l of 33 mM potassium phosphate buffer, pH 8.0, on ice overnight under anaerobic conditions, free DTT was removed from each sample with a NAP5 column before analysis.
A Target Residue of Oxidants-To investigate the protection of Cys 247 by oxidants against inactivation via carboxymethylation by iodoacetate (Nakalai Tesque), after incubation of 17 M MST with 17 M hydrogen peroxide or tetrathionate in 60 l of 20 mM potassium phosphate buffer, pH 7.4, on ice for 20 min, free oxidants were removed from each mixture and a control group without oxidation with a NAP5 column.
The enzyme-containing fractions were collected and concentrated to 60 l with a VIVASPIN. Iodoacetate was added to the two experimental groups with oxidation and a control group without oxidation at a concentration of 1 mM, and the mixtures were incubated on ice for 20 min. After gel filtration of each mixture with a NAP5 column, the enzymecontaining fractions were collected and concentrated to 40 l with VIVASPIN. 5 l of the mixture was incubated with 5 l of 2 mM DTT or the same buffer at 25°C for 20 min. The values of the rhodanese activity remaining were determined.
MALDI-TOF Mass Spectrometric Analysis for Oxidized MSTs-After 12 M MST was incubated with 12 M tetrathionate, 12 M hydrogen peroxide, or 0.6 mM hydrogen peroxide in 20 mM potassium phosphate buffer, pH 7.4, on ice for 20 min, each mixture was treated with gel filtration with a NAP5 column to remove free oxidants. For a control experiment, 12 M MST was incubated without tetrathionate or hydrogen peroxide, and the mixture was treated with gel filtration with a NAP5 column. Each enzyme-containing fraction was collected and concentrated to 60 l with VIVASPIN. For reduction of each oxidized enzyme, 30 l of each sample was treated with 0.6 mM DTT at 25°C for 20 min, and free DTT was removed with a NAP5 column. The enzymecontaining fractions were collected and concentrated to 20 l with a VIVASPIN.
To examine whether the hydrogen peroxide-inhibited MST was modified with dimedone (Tokyo Kasei Co. Ltd., Tokyo, Japan), 1.5 mM MST was incubated with 1.5 mM hydrogen peroxide in 20 mM potassium phosphate buffer, pH 7.4, on ice for 20 min. After gel filtration of the mixture with a NAP5 column, the enzyme-containing fractions were collected and concentrated to 36 l with a VIVASPIN. The solution was incubated with 50 mM dimedone on ice for 30 min. After gel filtration of the solution with a NAP5 column, the enzyme-containing fractions were collected and concentrated to 43 l with a VIVASPIN.
Each sample was desalted with Zip Tip C 18 (Millipore), and 0.5 l of the sample in 70% acetonitrile (Wako Pure Chemicals) containing 0.1% trifluoroacetic acid (Wako Pure Chemicals) was mixed with 0.5 l of sinapinic acid (Bruker Daltonics, Brumen, Germany) saturated in 50% acetonitrile containing 0.1% trifluoroacetic acid. The mixture was dried at room temperature on the target plate.
MALDI-TOF mass spectrometry was performed on a Reflex III (Bruker Daltonics) mass spectrometer equipped with a SCOUT 384 ion source laser, operating in the linear positive mode at a 20-kV acceleration voltage. Mass spectra were obtained by averaging 300 individual laser shots. External mass calibration was performed using the protein mixture of protein calibration standard II (Bruker Daltonics). In the mass spectrometric analysis of a chemical modification of 30 -40 of kDa protein using our system, mass measurement error was 0.03%.
Peroxidase Activity of MST-The assay mixture contained 0.2 mM NADPH, 40 M recombinant E. coli thioredoxin, and 1 M recombinant E. coli thioredoxin reductase in 500 l of 50 mM potassium phosphate buffer, pH 7.4. With monitoring the decrease in absorbance at 340 nm, 10 M hydrogen peroxide was added to the mixture, and 300 min later, 10, 20, 40, or 60 M wild type MST was added. The rate of decrease in NADPH was calculated (⑀ 340 ϭ 6220 M Ϫ1 ). For a control study, C247S was used instead of wild type MST.
Assay for Rhodanese Activity of MST-A procedure to measure the activity catalyzing a transsulfuration from mercaptopyruvate to ␤-mercaptoethanol would not be appropriate for this experiment, because ␤-mercaptoethanol would be able to reduce a sulfenate or a sulfenyl thiosulfate formed at a catalytic site cysteine during incubation in the assay mixture, as MST possesses rhodanese activity catalyzing transsulfuration from thiosulfate to cyanide (10,11) and it is this rhodanese activity of MST that was measured.
On the other hand, the amount of thiosulfate that is a derivative of tetrathionate was negligible in the rhodanese activity assay because only a very small amount of the compound was diluted further in the assay mixture.
Protein Determination-The protein concentrations were determined with a Coomassie protein assay kit (Pierce Biotechnology) with crystalline bovine serum albumin (ICN Biochemicals) as the standard.
Statistical Analysis-All values are expressed as the mean Ϯ the S.E. The significance of difference between values was estimated with Student's t test.
Data Fitting-The fitting of the data obtained from the kinetic studies was done with Kaleidagraph (Synergy Software, Reading, PA).
The activity of wild type MST was restored by in a time-dependent manner, and the time course is formulated as y ϭ 90.8 ϩ 10.1 log t (where y is the percent of control enzyme activity, and t is incubation time in min). An excess molar dose of DTT restored the tetrathionateinhibited MST activity faster than hydrogen peroxide-inhibited one.
These findings suggest that the redox potentials are different among the tetrathionate-and hydrogen peroxide-inhibited MSTs. The data suggest that a disulfide bond is not formed at the catalytic site Cys 247 , but rather a sulfenate is formed.
Reactivation of Oxidant-inhibited Wild Type MST by Glutathione, Thioredoxin, and Cysteine-The values of the activity of the hydrogen peroxide-inhibited and tetrathionate-inhibited wild type MSTs were restored by incubation with 1.6 mM DTT to 89.8 Ϯ 6.6 and 94.4 Ϯ 5.2% (n ϭ 3) of the control, respectively for 20 min (Fig. 2).
Thioredoxin together with the reducing system completely restored the values of the activity of hydrogen peroxide-and tetrathionate-inhibited MSTs to 106.2 Ϯ 9.8 and 113.6 Ϯ 8.6% (n ϭ 3) of each control value, respectively (Fig. 2). Furthermore, reduced thioredoxin restored activity to 91.5 Ϯ 6.3 and 98.6 Ϯ 7.2% of each control value, respectively. These results showed that these oxidized MSTs differ in terms of their redox potential, and the mid-redox potential of the hydrogen peroxide-inhibited MST was close to that of thioredoxin and lower than that of glutathione.
SH Group Titration and the Target Residue of Oxidants-The results of SH titration of the reduced and oxidized MSTs using DTNB and NBD-Cl (TABLE ONE) determined that Cys 154 , Cys 247 , and Cys 263 were exposed cysteines. Cys 154 and Cys 263 were outside cysteines, which was estimated from the data of the ternary structure of Leishmania MST (9), and partly contributed to a dimer formation via a disulfide bond. Iodoacetate inactivated MST (TABLE TWO) via carboxymethylation of Cys 247 . When MST had been inhibited by a stoichiometric concentration of hydrogen peroxide or tetrathionate prior to the treatment with iodoacetate, the activity was restored by DTT (TABLE TWO). These findings suggested that oxidants protect a catalytic Cys 247 against inactivation by iodoacetate, and therefore, Cys 247 is a target of oxidants. However, Cys 247 of the hydrogen peroxide-inhibited MST was not modified by NBD-Cl or DTNB, and no spectrophotometric change at 237 nm was observed. These findings show that the sulfenate in this case was not modified by NBD-Cl.
The  (Fig. 3). The difference in mass number (104 amu) was consistent with that of a thiosulfate (ϪSSO 3 Ϫ ) (32,890.6 Ϯ 9.9 (the expected mass number Ϯ mass measurement error)), suggesting that a sulfenyl thiosulfate was formed at Cys 247 by incubation with tetrathionate.
Peroxidase Activity of MST-When 10 M hydrogen peroxide was added to the assay mixture containing 0.2 mM NADPH, 40 M thioredoxin and 1 M thioredoxin reductase, the rate of consumption of NADPH was 3.78 M/min (Fig. 4); electrons were transferred from reduced thioredoxin directly to hydrogen peroxide (a scheme in Fig. 4).
In the presence of 10, 20, 40, or 60 M MST, the rate was increased to 2.65, 3.98, 6.72 (Fig. 4), or 7.96 M/min, suggesting that the peroxidase reaction proceeded in a MST concentration-dependent manner. Further, this reaction also proceeded in a hydrogen peroxide concentrationdependent manner (data not shown). Electrons were passed from  reduced thioredoxin to hydrogen peroxide via MST (a scheme in Fig. 4).
On the other hand, C247S did not possess the peroxidase activity (data not shown).

DISCUSSION
A sulfenyl compound and a sulfenate formed at the cysteine residues in proteins or enzymes (12-19, 21, 22) are stable in the absence of a nearby cysteine (21,22) or in a hydrogen bond-rich environment (21,22). A sulfenyl thiosulfate in tetrathionate-inhibited MST and a cysteine-sulfenate in hydrogen peroxide-inhibited MST were stable on ice for at least 48 h, indicating that there was no cysteine residue close to Cys 247 (data not shown). This was confirmed by DTNB and NBD-Cl titration of the SH-group.
In a rare case, protein-tyrosine phosphatase IB formed a sulfenyl amide with a main chain nitrogen atom of the adjacent amino acid (15,16). Replacement of the adjacent amino acid, Gly 248 with Arg or Ser 249 with Ala in rat MST did not affect inhibition kinetics (data not shown), indicating that a sulfenyl amide was not formed in this case. 3 Rhodanese (34) and NADH peroxidase (33,35) catalyzed the thioredoxin oxidase and peroxidase reaction, respectively, which strongly suggested cysteine-sulfenate formation. MST also possessed thioredoxin peroxidase activity only when the concentration of thioredoxin reductase was less than 1/20 that of MST. The peroxidase reaction should proceed via a sulfenic intermediate of thioredoxin (34).
A sulfenyl cysteine-NBD adduct was successfully detected in oxidized forms of NADPH oxidase (20), alkyl hydroperoxide reductase (23), ␣ 1 -antitrypsin (41), and serum albumin (28). The spectral property of sulfenate-NBD adducts showed maximal absorption at 347 nm, which  was different from that of free NBD-Cl at 343 nm (344 nm in this study) and the cysteine-NBD adduct at 420 nm (416 nm in this study). On the other hand, a spectral change at 347 nm was not observed in the hydrogen peroxide-inhibited MST after incubation with NBD-Cl, even when the sample was treated with 5 M guanidine hydrochloride or 8 M urea. Other possible reagents for modification of a sulfenate, 2-nitro-5-thiobenzoic acid (23, 26) and 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, were also not able to modify Cys 247 of the hydrogen peroxide-inhibited MST (data not shown). A MALDI-TOF mass spectrometric analysis revealed that the hydrogen peroxide-inhibited MST increased in mass number in a manner consistent with the formation of a sulfenate (-SO 3 Ϫ ), and DTT decreased the mass to that of control MST. An excess molar dose of hydrogen peroxide increased the molecular mass of MST consistent with that of the formation of a sulfonate (-SO 3 Ϫ ), and this mass was not significantly changed by DTT. This is consistent with the fact that DTT could not restore the activity. A crystallographic study of Leishmania MST confirmed a sulfonate formation at the catalytic cysteine (42). A peak indicating MST-sulfinate was not distinctively observable, probably because of being a minor constituent in oxidized MST species. It is noteworthy that reduced thioredoxin or thioredoxin along with the reducing system completely restored the activity of the tetrathionate-and hydrogen peroxide-inhibited MSTs. An excess molar dose of cysteine partly restored tetrathionate-inhibited MST, which is consistent with the finding that cysteine restored the activity of tetrathionateinhibited streptococcal proteinase via the formation of a sulfenyl thiosulfate at the catalytic cysteine (25). On the other hand, reduced glutathione or glutathione along with the reducing system partly restored the activity of tetrathionate-and hydrogen peroxide-inhibited MSTs. These findings suggest that the mid-redox potential of the cysteine-sulfenate in this case could be estimated as close to and lower than that of glutathione (Ϫ240 mV (43)) and higher than that of thioredoxin (Ϫ270 mV (43)).
In previous studies on the reduction of a cysteine-sulfenate, glutathione was used as an effective reductant (29 -32), meaning that the midredox potential of a cysteine-sulfenate was higher than that of glutathione. On the other hand, the mid-potential of a MST-sulfenate was exceptionally close to and lower than that of glutathione, and the characteristics of the MST-sulfenate were different from those reported previously. The stability and reactivity of a sulfenate contained in an enzyme has not been studied precisely. It has been only reported that a well developed network of hydrogen bonding interactions stabilizes the sulfenate (21,22,44). The lower reactivity of the MST-sulfenate with NBD-Cl and dimedone is probably because of this reported well developed network of hydrogen bonding (42) interlacing with it.
Cytosolic and mitochondrial MST (45) plays physiological role in the protection against oxidative stress, and peculiarly contributes to the maintenance of cellular redox homeostasis via the metabolic regulation of cysteine degradation.