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J. Biol. Chem., Vol. 279, Issue 48, 50025-50030, November 26, 2004

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The Yeast Prion Protein Ure2 Shows Glutathione Peroxidase Activity in Both Native and Fibrillar Forms^*

Ming Bai, Jun-Mei Zhou¶, and Sarah Perrett¶

From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China and Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Fengtai District, Beijing 100039, China

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, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterization of the purified protein. Here we use steady-state kinetic methods to characterize 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 behavior 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 wild type. Fibrillar aggregates showed the same level of activity as native 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.

Received for publication, June 14, 2004 , and in revised form, September 10, 2004.

^* This work was supported by the Natural Science Foundation of China (30070163, 30470363), the 973 Project of the Chinese Ministry of Science and Technology (G1999075608), and the Chinese Academy of Sciences Knowledge Innovation Project (KSCX2-SW214-3). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a special grant from the Chinese Academy of Sciences and research fellowships from the Royal Commission for the Exhibition of 1851 and the Royal Society. To whom correspondence may be addressed. Tel.: 86-10-64888496; Fax: 86-10-64840672; E-mail: sarah.perrett{at}iname.com or zhoujm{at}sun5.ibp.ac.cn.

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