Role of Amino Acid Residues in the Active Site of Rat Liver Mercaptopyruvate Sulfurtransferase

Abstract

A complete amino acid structure of rat liver mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 296 amino acid residues with a calculated molecular mass of 32,808 Da. Sequence identity in cDNA and the deduced amino acid sequence are 65 and 60% respectively, between rat MST and rhodanese. By their entire sequence similarity MST and rhodanese are confirmed to be evolutionarily related enzymes (Nagahara, N., Okazaki, T., and Nishino, T. (1995) J. Biol. Chem. 270, 16230 -16235). The conversion of MST to rhodanese was attempted, and the role of amino acid residues was studied by site-directed mutagenesis with the isolated cDNA of rat liver MST. In the reaction the outer sulfur atom of thiosulfate is transferred to the Cys residue of the enzyme molecule to form a persulfide intermediate, which is subsequently attacked by cyanide anion to give thiocyanide. However, the enzyme has rather wide substrate specificity. Certain other sulfur compounds such as thiosulfonate (2) or persulfides (3, 4) may substitute for thiosulfate; and sulfite (5), sulfinates (5), or various thiol compounds (6) may substitute for cyanide. This enzyme was reported to be widely distributed in prokaryote and eukaryote mitochondria (7-9). Although the physiological role of this enzyme is not well understood, the enzyme is well characterized. The enzyme was first isolated from bovine liver (10) and subsequently from rat liver (11). Primary structures of the enzyme from various sources were determined from protein or deduced from cDNA, e.g. bovine liver (12) and adrenal (13), chicken liver (14), human liver (15), rat liver (16), hamster ovary (17), and mouse liver (18). Further, recombinant bovine liver (13) and adrenal (19), rat liver (20), hamster ovary (17), and mouse liver (18) rhodanese were overexpressed in Escherichia coli and characterized. The enzyme from bovine liver was crystallized (10, 21), and the three-dimensional structure was determined. (22)(23)(24). On the other hand, mercaptopyruvate sulfurtransferase (MST, 1 EC 2.8.1.2) catalyzes the following reaction.The enzyme was discovered in rat liver quite a long time ago (25-28), but compared with rhodanese the enzyme was not well characterized. Although the enzyme responsible for this reaction was at first assumed to be rhodanese, subsequent investigation with crystallized rhodanese showed that rhodanese did not catalyze this reaction and that another enzyme, mercaptopyruvate sulfurtransferase, was responsible for the reaction (28). Similar to rhodanese, compounds other than cyanide may function as sulfur acceptors in the reaction catalyzed by this enzyme (29 -32), and its physiological role is a matter of discussion (33, 34). This enzyme was reported to be distributed in both prokaryotes and eukaryotes and to be located in the cytosol of eukaryotic cells (26,27, 34,35), but the existence of this enzyme in mitochondria is also reported (36). Recently rat liver MST was purified to homogeneity, and th...

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

“…This substrate preference correlates well with the active-site residues being homologous to other eukaryotic MSTs rather than rhodaneses. The K m and V max values obtained compare favorably with those reported for bacterial, plant, and vertebrate MSTs (1,42). The very high activity toward 3-mercaptopyruvate suggests that this could function as a natural substrate, although currently, there is nothing known about the levels of this compound in Leishmania parasites or any roles that it may have.…”

“…Eukaryotic MSTs can be cytosolic or mitochondrial (22,42,46). Both the immunolocalization and fractionation data suggest that the MST of L. major occurs within the cytosol.…”

Thioredoxin-dependent Enzymatic Activation of Mercaptopyruvate Sulfurtransferase

“…nol would not be appropriate for this experiment, because ␤-mercaptoethanol reduces disulfide bonds formed between subunits during incubation in the assay mixture. MST possesses rhodanese activity, which catalyzes trans-sulfuration from thiosulfate to cyanide (11,18); therefore, it was this rhodanese activity of MST that was measured.…”

“…Rat 3-mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2) is a 32.8-kDa simple protein enzyme (11). MST catalyzes the degradation of cysteine, detoxifies cyanide (12), and serves as anti-oxidant protein (13).…”

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…The MST protein is located in the cytosol and in the mitochondria of the eukaryotic cell (Wood and Fiedler, 1953;Meister et al, 1954;Westley, 1973;Nagahara et al, 1999). The enzyme was purified to homogeneity (Nagahara et al, 1995) and cloned from rat liver (Nagahara and Nishino, 1996). Both ST proteins, MST and rhodanese, isolated from the same organism (Rattus) accepted 3-MP and thiosulfate, but the ratios of their respective enzyme activities differed.…”