Lin Hsia-lien, Kent Ute M, Zhang Haoming, Waskell Lucy, Hollenberg Paul F
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Chem Res Toxicol. 2003 Feb;16(2):129-36. doi: 10.1021/tx020040b.
We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225. This peptide contains two tyrosine residues at positions 190 and 203. In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1. All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized. The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT. The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A. The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein. Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A. Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1. Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149.
我们之前曾报道，细胞色素P450 2B1会被过氧亚硝酸酯灭活，且催化活性的降低与酪氨酸硝化作用的增强相关。用过氧亚硝酸酯处理过的P450 2B1经Lys C消化，随后对含硝基酪氨酸的主要肽段进行氨基酸测序，结果表明该肽段跨越第160 - 225位残基。此肽段在第190和203位含有两个酪氨酸残基。在本研究中，我们将野生型重组P450 2B1（WT）中的Tyr 190突变为Ala（Y190A），并将Tyr 203突变为Ala（Y203A），以确定被硝化的特定残基，并确定硝基酪氨酸的形成是否是过氧亚硝酸酯介导的P450 2B1失活的原因。所有三种P450均在大肠杆菌中表达，纯化至同质，并进行了表征。与WT相比，P450 2B1的四种不同底物的催化活性对于Y203A突变体增加了约2倍，但对于Y190A突变体降低了约60%。向P450中加入过氧亚硝酸酯导致WT和Y203A的催化活性呈浓度依赖性降低，但Y190A的催化活性没有损失。与WT或Y203A蛋白相比，过氧亚硝酸酯对Y190A的酪氨酸硝化程度降低了约75%。用过氧亚硝酸酯修饰的蛋白经Lys C消化后，从WT和Y203A中检测到一个主要的含硝基酪氨酸的肽段，但从Y190A中未检测到。总体而言，这些结果表明Tyr 190是过氧亚硝酸酯介导的硝化作用的靶残基，并且该酪氨酸的硝化是P450 2B1失活的原因。模型研究表明，Tyr 190可能通过与Glu 149形成氢键，在维持蛋白质完整性以实现最大活性方面发挥结构作用。
