Nitration and inactivation of tyrosine hydroxylase by peroxynitrite
Nitration and inactivation of tyrosine hydroxylase by peroxynitrite
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DOI：
10.1074/jbc.m105564200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
Ischiropoulos, H
中科院分区：
文献类型：
--
作者：
Blanchard-Fillion, B;Souza, JM;Ischiropoulos, H
小鼠暴露于1-甲基-4-苯基-1，2，3，6-四氢苯基吡啶后，酪氨酸羟化酶（TH）被硝化修饰。酪氨酸硝化与体外TH活性失活的时间关联表明，这种共价翻译后修饰是体内TH功能丧失的原因（Ara，J.，Przedborski，S.，Naini，A. B.，杰克逊-刘易斯，V，特里菲莱蒂河R.，Horwitz，J.，和Ijuropoulos，H.等人（1998）Proc.美国科学院联合S. A. 95，7659 - 7663）。最近的数据表明，半胱氨酸氧化而不是酪氨酸硝化是导致体外过氧亚硝酸盐暴露后TH失活的原因（Kuhn，D. M.，艾瑞莎，C。W.，和Geddes，T. J.（1999）J. Neurosci. 19，10289-10294）。然而，重新检查过氧化亚硝酸盐与纯化TH的反应未能产生半胱氨酸氧化，但导致浓度依赖性增加酪氨酸硝化和失活。半胱氨酸氧化仅在蛋白质部分解折叠后观察到。酪氨酸残基423和较小程度的酪氨酸残基428和432通过硝化被修饰。Tyr（423）突变为Phe导致与野生型蛋白质相比硝化作用降低而活性不损失。停流实验表明，在pH7.4和25 ℃下，过亚硝酸根与TH反应的二级速率常数为（3.8 ± 0.9）× 10（3）M ~（-1）s ~（-1）。总的来说，数据表明，过氧亚硝酸盐与蛋白质的金属中心反应，并主要导致酪氨酸残基423的硝化，这是负责TH的失活。
Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad Sci. U. S. A. 95, 7659 -7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) X 10(3) M-1 s(-1) at pH 7.4 and 25 degreesC for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.