Role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase. CDNA cloning, overexpression, and site-directed mutagenesis.
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. There is a strong possibility that Cys247 is a catalytic site of MST. Arg187 is suggested to be a binding site of both mercaptopyruvate and thiosulfate in MST. Arg196, which is missed in rhodanese, is important for catalysis in MST. On the other hand, the substitution of Arg for Gly248 or Lys for Ser249 facilitates catalysis of thiosulfate in MST. It is concluded that Arg187 and Arg196 of rat MST are critical residues in determining substrate specificity for mercaptopyruvate. On the other hand, Arg185, Arg247, and Lys248 of rat rhodanese are critical residues in determining substrate specificity for thiosulfate.
Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi Bunkyo-ku, 113 Tokyo, Japan.
N
N
Nagahara
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T
Nishino
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013466:Sulfurtransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C021550:3-mercaptopyruvate sulphurtransferase; D003545:Cysteine