Site directed mutagenesis of Schizosaccharomyces pombe glutathione synthetase produces an enzyme with homoglutathione synthetase activity.

ABSTRACT: Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k(m) 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (k(m) 0,07) and lowered the affinity for Gly (k(m) 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k(m) 0,23) as a substr

SUBMITTER: Dworeck T 

PROVIDER: S-EPMC3473041 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature