TY - JOUR
T1 - Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid
AU - Yang, Kap Seok
AU - Kang, Sang Won
AU - Woo, Hyun Ae
AU - Hwang, Sung Chul
AU - Chae, Ho Zoon
AU - Kim, Kanghwa
AU - Rhee, Sue Goo
PY - 2002/10/11
Y1 - 2002/10/11
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037064080&partnerID=8YFLogxK
U2 - 10.1074/jbc.M206626200
DO - 10.1074/jbc.M206626200
M3 - Article
C2 - 12161445
AN - SCOPUS:0037064080
SN - 0021-9258
VL - 277
SP - 38029
EP - 38036
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -