Journal of Biological Chemistry
Volume 277, Issue 41, 11 October 2002, Pages 38029-38036
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ENZYME CATALYSIS AND REGULATION
Inactivation of Human Peroxiredoxin I during Catalysis as the Result of the Oxidation of the Catalytic Site Cysteine to Cysteine-sulfinic Acid*

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By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys51-SH to Cys51-SO2H. Thus, Cys51-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys51-SO2H, which cannot be reversed by thioredoxin. The presence of H2O2 alone was not sufficient to cause oxidation of Cys51 to Cys51-SO2H. Rather, the presence of complete catalytic components (H2O2, thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys172/Ser172 mutant Prx I required not only H2O2, but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 μm) of H2O2 indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 °C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H2O2.

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Published, JBC Papers in Press, August 2, 2002, DOI 10.1074/jbc.M206626200

*

This work was supported in part by Korea Research Foundation Grant KRF-2001-015-DP0482 (to H. Z. C.) and by the Korea Science and Engineering Foundation through the Center for Cell Signaling Research at Ewha Womans University, Korea (to H. A. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

§

Present address: Center for Cell Signaling Research and Div. of Molecular Life Sciences, Ewha Womans University, 11-1 Daehyun Dong, Seodaemoon-gu, Seoul 120-750, Korea.

On leave from the Dept. of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, Suwon 442-749, Korea.

On leave from the Dept. of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 500-757, Korea.

**

Present address: Dept. of Food and Nutrition, College of Home Economics, Chonnam National University, Gwangju 500-757, Korea.