Glutathione transferases : molecular cloning, site-directed mutagenesis and structure-function studies

Sammanfattning: Three distinct glutathione transferases (GST) cDNA clones were obtained by screening of tumour cell line cDNA libraries. The cDNA clones were expressed in Escherichia coli and the enzymes characterized. Rat GST 8-8 is the only member in the multifunctional GST family for which a specific cellular function has been proposed. Several new substrates among toxic unsaturated compounds were found, supporting the idea that rat GST 8-8 is involved in the cellular protection against oxidative stress.A partial cDNA clone encoding human class Pi GST was extended with synthetic DNA, designed to optimize expression in E. coli. A ten-fold increased yield of protein was obtained as compared to previous expression constructs.Human GST Al-1 was subjected to site-directed mutagenesis for structure-function analysis. A tyrosine residue was identified as an important component of the active site, by demonstrating that a Tyr7—>Phe mutant displayed strongly reduced catalytic activity and affinity for GSH.The role of evolutionary conserved arginine residues for structural stability and catalytic function was investigated, by construction of four Arg—> Ala mutants. The results demonstrate that three of these arginine residues are important for maintaining a funtional enzyme. The results are discussed in relation to a recently determined 3D structure of a pig lung GST.