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 ( ) inhibited rat MST (k_i = 3.3 min^–1, K_i = 120.5 μm and k_i = 2.5 min^–1, K_i = 178.6 μm, 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 Cys²⁴⁷, suggesting that Cys²⁴⁷ is a target of the oxidants. A matrix-assisted laser desorption/ionization–time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate ( ), 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 Cys²⁴⁷, 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.

Human mercaptolactate-cysteine disulfiduria (MCDU) was first recognized and reported in 1968. Most cases of MCDU are associated with mental retardation, while the pathogenesis remains unknown. To investigate it, we generated homozygous 3-mercaptopyruvate sulfurtransferase (MST: EC 2.8.1.2) knockout (KO) mice using C57BL/6 embryonic stem cells as an animal model. The MST-KO mice showed significantly increased anxiety-like behaviors with an increase in serotonin level in the prefrontal cortex (PFC), but not with abnormal morphological changes in the brain. MCDU can be caused by loss in the functional diversity of MST; first, MST functions as an antioxidant protein. MST possessing 2 redox-sensing molecular switches maintains cellular redox homeostasis. Second, MST can produce H₂S (or HS⁻). Third, MST can also produce SO_x. It is concluded that behavioral abnormality in MST-KO mice is caused by MST function defects such as an antioxidant insufficiency or a new transducer, H₂S (or HS⁻) and/or SO_x deficiency.

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.

Rat 3‐mercaptopyruvate sulfurtransferase (MPST) is a 32 808 Da simple protein. Cys²⁴⁷ is a catalytic site, and Cys¹⁵⁴ and Cys²⁶³ are on the enzyme surface. MPST is found in all tissues, particularly in the kidneys, although the localization of its activity differs in each tissue. In this review, four functions of MPST are reviewed: (i) antioxidative function: Cys²⁴⁷ is redox‐sensitive and serves as a redox‐sensing switch. It is oxidized to cysteine sulfenate, which has a low redox potential, upon which the enzyme is inactivated. Then, reduced thioredoxin (Trx) with a reducing system (Trx reductase and NADPH) reduces the sulfenate to restore activity; meanwhile, Cys¹⁵⁴ and Cys²⁶³ form an intermolecular disulfide bond, which serves as another redox‐sensing switch. Consequently, Trx specifically cleaves the intermolecular disulfide bond by converting it from the inactive form (dimer) to the active form (monomer). (ii) Hydrogen sulfide and polysulfide production: hydrogen sulfide is produced via reduction of the persulfurated sulfur‐acceptor substrate by reduced Trx or Trx with a reducing system; as an alternative process, stable polysulfurated or persulfurated Cys²⁴⁷ as a reaction intermediate is reduced by Trx with a reducing system to release hydrogen sulfide and polysulfides. (iii) Possible sulfur oxide production: sulfur oxides (SO, SO₂ and SO₃) can be produced in the redox cycle of sulfane sulfur formed at the catalytic site Cys²⁴⁷ (Cys–SO⁻, Cys–SO²⁻ and Cys–SO³⁻) as reaction intermediates and released by reduced Trx or Trx with a reducing system. (iv) Possible anxiolytic‐like effects: MPST‐knockout mice exhibited anxiolytic‐like effects.