Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese. Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis.
Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity. MST is very similar to rhodanese in physicochemical properties. Further, rhodanese cross-reacts with anti-MST antibody. Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese. In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined. The sequence showed 66% homology with that of rat liver rhodanese. An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively. Conversion of rhodanese to MST was tried by site-directed mutagenesis. After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system. The mutation of these residues decreases rhodanese activity and increases MST activity.
Nagahara
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N
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Okazaki
T
T
Nishino
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D005075:Biological Evolution; D015153:Blotting, Western; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics*
D011994:Recombinant Proteins; C008366:3-mercaptopyruvic acid; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C021550:3-mercaptopyruvate sulphurtransferase; D003545:Cysteine