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 hom ogeneity. MST is very similar to rhodanese in physicochemical properti es. Further, rhodanese cross-reacts with anti MST antibody. Both purif ied authentic MST and expressed rhodanese possess MST and rhodanese ac tivities, although the ratio of rhodanese to MST activity is low in MS T and high in rhodanese. In order to compare the active site regions o f MST and rhodanese, the primary structure of a possible active site r egion of MST was determined. The sequence showed 66% homology with tha t of rat liver rhodanese. An active site cysteine residue (Cys(246); s ite of formation of persulfide in catalysis) and an arginine residue ( Arg(185); substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg(247), and Lys(248)) were replaced by Gly and Ser, respectively. Conversion of rh odanese to MST was tried by site-directed mutagenesis. After cloning o f rat liver rhodanese, recombinant wild type and three mutants (Arg(24 7) --> Gly and/or Lys(248) --> Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21(DE3) with a T7 promoter sys tem. The mutation of these residues decreases rhodanese activity and i ncreases MST activity.