Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes:: Characterization of reactive intermediate adducts to the heme and apoprotein
Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes:: Characterization of reactive intermediate adducts to the heme and apoprotein
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DOI：
10.1021/tx020052x
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发表时间：
2002-12-01
影响因子：
4.1
通讯作者：
Hollenberg, PF
中科院分区：
文献类型：
--
作者：
Blobaum, AL;Kent, UM;Hollenberg, PF
研究了细胞色素P450 2 E1和突变体P450 2 E1 T303 A在叔丁基乙炔（tBA）和叔丁基1-甲基-2-丙炔基醚（tBMP）作用下的失活动力学。这两种乙炔以时间、浓度和NADPH依赖性方式灭活纯化的兔P450 2 E1和2 E1 T303 A的7-乙氧基-4-（三氟甲基）香豆素（7-EFC）O-脱乙基活性。tBA对P450 2 E1和2 E1 T303 A的灭活Ki值分别为1.0和2.0 mM，k（inact）值分别为0.20和0.38 min（-1），t（1/2）值分别为3.5和1.8 min。tBMP失活的P450的K-I值分别为0.1和1.0 mM，k（inact）值分别为0.12和0.07 min（-1），t（1/2）值分别为5.9和10.2 min。酶活性的损失发生在P450 CO光谱和P450血红素，这是伴随着两个不同的tBA或tBMP-修饰的血红素产品在每个失活的样品中的外观的同时损失。加合物的LC-MS分析显示质量为661或705 Da，分别与铁耗尽的血红素的质量加上tBA或tBMP反应性中间体和一个氧原子的质量一致。只有tBA失活的P450 2 E1显示tBA加合的载脂蛋白，其质量增加99 Da，对应于tBA加一个氧原子的质量。令人惊讶的是，失活，CO光谱和血红素损失，和血红素加合物形成的tBA失活的T303 A突变体是完全可逆的透析后。此外，对硝基苯酚的代谢不受tBA失活的T303 A突变体的影响。因此，我们对tBA和tBMP对P450 2 E1和2 E1 T303 A的失活的研究表明存在三种不同的失活机制，其中包括一种新的、可逆的血红素烷基化。这是以前没有用P450酶描述过的。
The kinetics for the inactivation of cytochrome P450 2E1 and the mutant P450 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) were investigated. The two acetylenes inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified rabbit P450s 2E1 and 2E1 T303A in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. The K, values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the k(inact) values were 0.20 and 0.38 min(-1), and the t(1/2) values were 3.5 and 1.8 min, respectively. The K-I values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the k(inact) values were 0.12 and 0.07 min(-1), and the t(1/2) values were 5.9 and 10.2 min, respectively. Losses in enzyme activity occurred with concurrent losses in the P450 CO spectrum and P450 heme, which were accompanied by the appearance of two different tBA- or tBMP-modified heme products in each inactivated sample. LC-MS analysis of the adducts showed masses of 661 or 705 Da, consistent with the mass of an iron-depleted heme plus the masses of a tBA or tBMP reactive intermediate and one oxygen atom, respectively. Only the tBA-inactivated P450 2E1 revealed a tBA-adducted apoprotein with an increase in mass of 99 Da, corresponding to the mass of tBA plus one oxygen atom. Surprisingly, the inactivation, CO spectral and heme loss, and heme adduct formation of the tBA-inactivated T303A mutant were completely reversible after dialysis. In addition, metabolism of paranitrophenol was not compromised by the tBA-inactivated T303A mutant. Therefore, our studies on the inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMP suggest the existence of three distinct mechanisms for inactivation, among which includes a novel, reversible heme alkylation. that has not been previously described with P450 enzymes.