Kuhn Donald M, Sadidi Mahdieh, Liu Xiuli, Kreipke Christian, Geddes Timothy, Borges Chad, Watson J Throck
Department of Psychiatry, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 2002 Apr 19;277(16):14336-42. doi: 10.1074/jbc.M200290200. Epub 2002 Feb 7.
Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.
酪氨酸羟化酶（TH）是神经递质多巴胺生物合成过程中的起始和限速酶，可被过氧亚硝酸盐灭活。通过基质辅助激光解吸飞行时间质谱和酪氨酸扫描诱变技术，已确定了过氧亚硝酸盐诱导TH发生酪氨酸硝化的位点。用基质辅助激光解吸飞行时间质谱分析硝化TH的V8蛋白酶解片段。一个3135.4道尔顿的肽段，对应于TH的V410 - E436残基，显示出过氧亚硝酸盐诱导的质量位移分别为+45、+90和+135道尔顿，分别反映了一个、两个或三个酪氨酸的硝化。这些修饰在未处理的TH中不明显。将该肽段内的酪氨酸残基（第423、428和432位）突变为苯丙氨酸，以确认硝化位点并评估突变对TH活性的影响。单突变体表达野生型水平的TH催化活性，可被过氧亚硝酸盐灭活，同时显示出较低（30 - 60%）的硝化水平。双突变体Y423F、Y428F、Y423F、Y432F和Y428F、Y432F在暴露于过氧亚硝酸盐后显示出微量的酪氨酸硝化（为对照的7 - 30%），三突变体Y423F、Y428F、Y432F不是硝化的底物，但过氧亚硝酸盐显著降低了每个突变体的活性。当用PEO - 马来酰亚胺活化生物素（一种特异性标记蛋白质中还原型半胱氨酸残基的硫醇反应试剂）探测所有酪氨酸突变体时，很明显过氧亚硝酸盐导致了半胱氨酸氧化。这些研究确定了残基Tyr(423)、Tyr(428)和Tyr(432)是过氧亚硝酸盐诱导TH硝化的位点。没有单个酪氨酸残基对TH催化功能似乎至关重要，酪氨酸硝化对于过氧亚硝酸盐诱导的失活既非必要条件也非充分条件。过氧亚硝酸盐导致的TH催化活性丧失反而与半胱氨酸残基的氧化有关。
