“Restoration” of Glutathione Transferase Activity By Single-site Mutation of The Yeast Prion Protein Ure2

The yeast prion Ure2p is composed of an N-terminal prion domain, and a C-terminal globular domain, which shows similarity to glutathione transferases (GSTs) in both sequence and structure. Ure2p protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity. Ure2p shows glutathione-dependent peroxidase (GPx) activity, which is often an adjunct activity of GSTs, but wild-type Ure2p shows no detectable GST activity toward the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB). The structural basis for the substrate specificity of Ure2p enzymatic activity is an interesting problem that is fundamental to understanding the in vivo roles of Ure2p and its relationship to the GST structural family. The critical catalytic residue in the other known GSTs is Ser, Tyr or Cys. Here, we demonstrate that residue N124 is important for the GPx activity of Ure2p, and a wild-type level of activity is maintained in N124S, but not in N124Y/C. Interestingly, we found that the single-site mutations A122C and N124A/V (but not N124S/Y/C) ”restore” the GST activity of Ure2p toward CDNB, while causing a substantial reduction in GPx activity. The steady-state kinetics for the GST activity of A122C appears to follow a ping-pong mechanism. In contrast, the GST activity of 124-site mutants shows a sequential mechanism, as is observed for the native GPx activity of Ure2p, and typical GST enzymes. These findings shed light on the evolutionary relationship of Ure2p with other GST family members, and contribute to our understanding of catalytic promiscuity and divergent evolution.