The mercaptopyruvate pathway in cysteine catabolism: a physiologic role and related disease of the multifunctional 3-mercaptopyruvate sulfurtransferase.

The conversion of cysteine to 3-sulfino-alanine is a major pathway in cysteine catabolism. Cysteine dioxygenase catalyzes the reaction and dietary intake of the essential amino acid methionine and the semi-essential amino acid cysteine increases the level of this enzyme by suppressing enzyme degradation via polyubiquitination. The production of cellular antioxidants such as glutathione, thioredoxin, and their families is important in cysteine metabolism, and these cellular antioxidants have critical roles in the maintenance of the cellular redox status. The mercaptopyruvate pathway, in which cysteine or aspartate transaminase catalyzes the transamination from cysteine to 3-mercaptopyruvate and then 3-mercaptopyruvate sulfurtransferase catalyzes the transsulfuration from 3-mercaptopyruvate to pyruvate, also contributes to maintain the cellular redox. 3-Mercaptopyruvate sulfurtransferase serves as an antioxidant protein: when the enzyme is exposed to stoichiometric amounts of the oxidant hydrogen peroxide, it is inhibited via the formation of low redox sulfenate at the catalytic site cysteine. On the other hand, activity is restored by the reductant dithiothreitol or reduced thioredoxin. 3-Mercaptopyruvate sulfurtransferase also detoxifies cyanide via transsulfuration from a stable persulfide at the catalytic site cysteine, a reaction intermediate, suggesting that cyanide detoxification is not necessarily an enzymatic reaction. Furthermore, a congenital defect of the enzyme causes mercaptolactate-cysteine disulfiduria associated with or without mental retardation, although the pathogenesis remains unclear. These facts suggest that 3-mercaptopyruvate sulfurtransferase has physiologic roles as an antioxidant and a cyanide antidote; is essential for neural function, and participates in cysteine degradation.

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center.

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Cys(122), Cys(124) and Cys(132) are placed near two catalytic sites, Lys(199) and Lys(252), and coordinate a zinc ion, referred to as "a proximal zinc ion", and Cys(223) is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as "a distal zinc ion", with His(131) . When the wild-type enzymes C122A (Cys(122)-->Ala), C124A (Cys(124)-->Ala), C132A (Cys(132)-->Ala) and C223A (Cys(223)-->Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys(122), Cys(124) and Cys(132) under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys(122), Cys(124) and Cys(132) was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.

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.

Do antidotes for acute cyanide poisoning act on mercaptopyruvate sulfurtransferase to facilitate detoxification?

A well-known combined therapy for acute cyanide poisoning is intravenous administration of sodium nitrite, sodium thiosulfate and cobalt EDTA. Sodium nitrite oxidizes oxy-hemoglobin, resulting in methemoglobin, which has a high affinity for cyanide. Sodium thiosulfate is a substrate of thiosulfate: cyanide sulfurtransferase (rhodanese, EC.2.8.1.1), and facilitates catalytic metabolism of cyanide to less toxic thiocyanate. Cobalt EDTA combines with cyanide to reduce cyanide in the blood. Here, we focus on cytosolic and mitochondrial mercaptopyruvate sulfurtransferase (MST, EC. 2.8.1.2), which detoxifies cyanide more effectively than rhodanese, because rhodanese is localized only in mitochondria. Thiosulfate also serves as a substrate of MST and cyanide can be metabolized to thiocyanate. However, the K(m) value for thiosulfate is so large that it is not expected to contribute much to the detoxification of cyanide. On the other hand, nitrite and cobalt EDTA did not affect MST. Thus, combined therapy only slightly acts on MST to detoxify cyanide. Some investigators have attempted a new therapy in which mercaptopyruvate, a substrate of MST was administered intravenously, but this was not effective for detoxification due to the rapid decomposition of mercaptopyruvate in the blood. There are two possible strategies to facilitate MST activities: development of modified mercaptopyruvate with a longer half time and development of a chemical compound which indirectly increases transcription of MST via regulation of a DNA binding protein.