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Papers In Press, published online ahead of print September 15, 2004
National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing 100101
Corresponding Author: sarah.perrett{at}iname.com
Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homology to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, while prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterisation of the purified protein. Here we use steady-state kinetic methods to characterise the multisubstrate peroxidase activity of Ure2p, using GSH with cumene hydroperoxide, hydrogen peroxide or tert-butyl hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concentration and showed optima at pH 8 and 40°C. Michaelis-Menten behaviour with convergent straight lines in double reciprocal plots was observed. This excludes a ping-pong mechanism and implies either a rapid-equilibrium random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to WT. Fibrillar aggregates showed the same level of activity as soluble protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.
J. Biol. Chem, 10.1074/jbc.M406612200
Submitted on June 14, 2004
Revised on September 10, 2004
Accepted on September 15, 2004
The yeast prion protein Ure2 shows glutathione peroxidase activity in both native and fibrillar forms
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