Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.
Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.
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细胞色素 P450 2B4 的光亲和标记：通过光卡宾捕获活性位点血红素配体。
DOI：
10.1021/bi00169a023
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
White,RE
中科院分区：
文献类型：
--
作者：
Miller,JP;White,RE
摘要：Spiro [金刚烷-2，2 '-diazirine]在光解时产生金刚烷基卡宾，与P450 2B 4（Ks= 3.2 uM）紧密结合，给出正常底物结合差谱。在天然铁P450 2B 4存在下，在365 nm处照射2-[3 H]金刚烷二氮杂环丙烷导致一级光解（fi/2= 1.8 min）。主要产物为2-[3 H]金刚烷醇，约6%的放射性与P450 2B 4共价结合。随着羰基亚铁形式的P450 2B 4，2-金刚烷醇的生产减少，蛋白质标记增加到12%。当使用氰化铁2B 4时，除了2-金刚烷醇之外，还形成2-金刚烷甲腈。腈似乎是由于铁结合的氰化物配体被卡宾捕获而产生的。使用多个周期的光解将蛋白质标记的百分比增加到76%。已知的2B 4底物和抑制剂可抑制光标记。此外，A-去甲基化的苄非他明和产生的底物结合差异光谱的苄非他明都抑制化学计量与放射性标记的蛋白质的馏分。标记的蛋白质被永久转换为高自旋状态，如吸收光谱中的特征变化所示，表明金刚烷基残基不可逆地占据底物结合位点。在五个Asp-Pro键处对放射性标记的2B 4进行温和的酸水解，生成一个2 kDa的肽，其携带78%的放射性。这些结果被解释为活性位点卡宾通过三种竞争途径反应的结果：捕获血红素第六配体以产生2-金刚烷醇或2-金刚烷腈，捕获未结合的活性位点水分子以产生金刚烷醇，以及共价连接到蛋白质残基。因此，P450 2B 4活性位点似乎含有至少一个未结合的水分子，除了血红素水第六配体，即使当底物存在。
Revised Manuscript Received November 10, 1993® abstract: Spiro [adamantane-2, 2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (Ks= 3.2 uM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H] adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted infirst-order photolysis (fi/2= 1.8 min). The main product was 2-[3H] adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4substrates and inhibitors. Also, A-demethylation of benzphetamineand generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the fiveAsp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active siteappears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.