Chapter Thirteen - Redox Regulation of Mammalian 3-Mercaptopyruvate Sulfurtransferase
Introduction
3-Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was discovered in the rat liver (Meister, 1953, Wood and Fiedler, 1953), as a cystine-catabolizing enzyme involved in the mercaptopyruvate pathway (Nagahara & Sawada, 2006). MST is distributed throughout prokaryotes and eukaryotes (Jarabak and Westley, 1978, Meister, 1953, Wood and Fiedler, 1953). The enzyme was first obtained in its highly purified form from rat liver (Nagahara, Okazaki, & Nishino, 1995) and was first cloned by Nagahara and Nishino (1996). The tertiary structures of MSTs isolated from Escherichia coli (Spallarossa et al., 2004) and Leishmania major (Alphey, Williams, Mottram, Coombs, & Hunter, 2003) are known but that of mammalian MSTs have not been determined.
Rat MST consists of 296 amino acids (molecular mass, 32,808 Da) and is a simple protein enzyme (Nagahara & Nishino, 1996). The enzyme contains three exposed cysteines: one catalytic site cysteine, Cys247, and two cysteines on the surface of the enzyme, Cys154 and Cys263 (Fig. 1; Nagahara & Nishino, 1996). The cysteine corresponding to position Cys263 is conserved among mammalian MSTs; on the other hand, Cys154 is unique to murine MST.
The finding that MST is evolutionarily related to mitochondrial rhodanese is of importance; sequence identity in protein and cDNA between rat MST and rhodanese are 60% and 65%, respectively (Nagahara and Nishino, 1996, Nagahara et al., 1995). An MST motif sequence “GSG” follows the catalytic site cysteine of MST, and a rhodanese motif sequence “RKG” follows the catalytic site cysteine of rhodanese (Fig. 1; Nagahara and Nishino, 1996, Nagahara et al., 1995). Using site-directed mutagenesis of these amino acids, we succeeded in switching the catalytic activity of MST to that of rhodanese and vice versa (Nagahara and Nishino, 1996, Nagahara et al., 1995).
Structural features of the gene encoding the enzyme have also been identified (Nagahara and Nishino, 1996, Nagahara et al., 2004). Promoter activity assay and cap site hunting analysis for human MST (Nagahara et al., 2004) revealed that the promoter region has features of a typical promoter of a housekeeping gene, containing GC box and silencer element (Nagahara et al., 2004). We also showed that each point mutation in the silencer module (− 394G → T or C, − 393C → T, or − 392T → G; nucleotide A of initiation ATG is designated as + 1) markedly enhanced the silencing function and reduced translation of MST (Nagahara et al., 2004). It is interesting to note that, MST expressions in the mouse brain and lung were increaed on embryonic Day 14 and were identical or slightly decreased in postnatal period (Fig. 2).
MST catalyzes a trans-sulfuration reaction in which the sulfur of mercaptopyruvate or thiosulfate is transferred to thiol-containing compounds or cyanide (Nagahara and Nishino, 1996, Nagahara et al., 1995). Km values for mercaptopyruvate and thiosulfate are 1.2 and 73 mM, respectively (Nagahara et al., 1995). During the catalytic process on the rat enzyme, Arg187 at the orifice of the active center serves as a binding site of the substrate, mercaptopyruvate, and a critical residue for catalysis. The positively charged nitrogen of the side chain of Arg187 interacts with the oxygen of the carbonyl group of mercaptopyruvate (referred as to nucleophilic assistance; Nagahara & Nishino, 1996).
The catalytic site cysteine is redox active and the pKa (approximately 6.0 in rat MST) is lower than that of free cysteine. After substrate binding, a stable persulfide is formed at the catalytic site cysteine as a reaction intermediate. It is noteworthy that the persulfide is stable at 4 °C, for at least 1 month. In rat MST, hydrogen bonds between the outer sulfur of the persulfide at Cys247 and Ser248 and between the outer sulfur of the persulfide at Cys247 and Thr252 may stabilize the persulfide (deduced by the results in Leishmania MST, Alphey et al., 2003). We recently recognized that the stable persulfide could serve as a reservoir of sulfur atom for subsequent formation of hydrogen sulfide.
The reactions involved in MST catalysis are shown in Fig. 3 (Nagahara, Ito, & Minami, 1999). When cyanide attacks the persulfide, less toxic thiocyanate is formed. This reaction is thought to detoxify cyanide. When thiol-containing compounds attack the persulfide, a new persulfide molecule is formed which can be subsequently reduced to hydrogen sulfide by thioredoxin (Fig. 3; Shibuya et al., 2009, Yadav et al., 2013). Polysulfides are usually formed during the sulfuration reaction (Fig. 3; Kimura, 2013). If thioredoxin serves as a sulfur, thioredoxin persulfide is formed which can be subsequently reduced to hydrogen sulfide by thioredoxin (Fig. 3; Yadav et al., 2013). A single substrate reaction occurs and hydrogen sulfide can be produced (Fig. 3).
MST is found in all the tissues in rat; however, its activity differs in each tissue (Nagahara, Ito, Kitamura, & Nishino, 1998). The specific activity is highest in the kidney, followed by that in liver and the heart, findings supported by northern blot analysis (Nagahara et al., 1998). Immunohistochemical analysis using a laser confocal microscopy have shown that the MST is localized in the proximal renal tubular cells in the kidney, pericentral hepatic cells in the liver, myocardial cells in the heart, perivascular glial cells in the brain, and bronchiolar epithelial cells in the lung (Nagahara et al., 1998).
Subcellular fractionation analysis revealed that eukaryotic MST activity was observed in both the cytoplasm and mitochondria with the specific activities of 1.39 ± 0.05 and 5.62 ± 0.15 units/mg, respectively (Nagahara et al., 1998, Nakamura et al., 2000). The findings were supported by Western blot analysis and immunoelectron microscopy study (Nagahara et al., 1998). Cytoplasmic and mitochondrial enzyme molecules are identical; however, a mitochondrial transport mechanism for MST has not yet been identified.
MST activity is regulated by redox change (Nagahara, 2008, Nagahara, 2013, Nagahara and Katayama, 2005, Nagahara et al., 2007). Under oxidizing conditions, MST is reversely inhibited to increase the cysteine pool, resulting in increase of cellular reductants thioredoxin, glutathione, or glutaredoxin. Thus, MST partly contributes to maintaining redox homeostasis and serves as an antioxidative protein.
Mercaptolactate-cysteine disulfiduria is a genetic disease known to be caused by abnormal MST activity. These patients show insufficiency or deficiency of MST. The symptoms include oversecretion of mercaptolactate-cysteine disulfide in the urine, with or without mental retardation (Crawhall et al., 1968). Due to MST deficiency, mercaptopyruvate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) to produce mercaptolactate. Mental retardation might be due to incomplete development of the brain. The function of antioxidative agents, hydrogen sulfide production, and/or sulfur oxides production (described below) might also be impaired at specific stages of embryonic development. However, the mechanism underlying functional abnormality in the brain has not been identified.
Section snippets
Redox Regulation of Cysteine Metabolism and MST
The cysteine pool in cells is increased under oxidizing conditions mainly because of post-translational inhibition of methionine synthase (EC 2.1.1.13; Mosharov et al., 2000, Nagahara and Sawada, 2006, Taokam et al., 1998) and post-translational activation of cystathionine β-synthase (EC 4.4.1.1; Fig. 4; Chen et al., 1995, Nagahara and Sawada, 2006). As a result, de novo synthesis of reductants, such as glutathione, thioredoxin, and/or glutaredoxin, is facilitated. Further, the transcription of
Redox-sensing molecular switches
Redox-sensitive cysteines in a switch-carrying protein such as enzymes, transcriptional factors, transcriptional factor modulators, receptor proteins, and sensor proteins serve as redox-sensing molecular switches. These switches can be classified into intermolecular and intramolecular types (Nagahara, 2011a, Nagahara, 2011b, Nagahara, 2012, Nagahara, 2013). Further, these switches can be classified into three subtypes: thioredoxin-, glutathione-, and glutaredoxin-specific, depending on reducing
MST Knockout Mouse
We produced MST-knockout mice to investigate the pathogenesis of mercaptolactate-cysteine disulfiduria and to determine the biological function such as antioxidative function of MST in the embryonic and postnatal stages. Some organs in the mouse cannot be defensed against oxidative stress due to the impairment of regulation of MST activity.
Western blot analysis confirmed that there was no expression of MST (Nagahara et al., 2013) in the knockout mice. No morphological changes were observed;
Other Investigation
Recently, we indirectly demonstrated production of sulfur oxides (SOx) during redox cycle of the persulfide (reaction intermediate) formed at the catalytic cysteine of MST, using MALDI-TOF mass spectrometry (Fig. 16; Nagahara et al., 2012). The persulfide at Cys247 is oxidized to thiosulfenyl MST (MST–SSO−), thiosulfinyl MST , and thiosulfonyl MST and MST is inhibited. Reducing agents can reduce them to form sulfur oxides. Interestingly, cyanide can also release sulfur oxides
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