Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1.
Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1.
复制标题
9-乙炔基菲灭活细胞色素 P450 2B1 的机理研究。
DOI：
10.1006/abbi.1995.9960
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发表时间：
1995
影响因子：
3.9
通讯作者：
Hollenberg,PF
中科院分区：
文献类型：
--
作者：
Roberts,ES;Ballou,DP;Hopkins,NE;Alworth,WL;Hollenberg,PF
本文研究了9-乙炔基菲（9-ethynylphenanthrene，9 EPh）对大鼠肝细胞色素P450 2B 1的失活机理。基质辅助激光解吸电离-质谱分析溴化氰产生的肽从9 EPh-灭活的P450 2B 1证实了菲乙酰基基团添加到对应于残基290至314的肽。当用胃蛋白酶进一步消化该肽时，可以将连接位点指定为肽Phe 297至Leu 307中的氨基酸之一[Roberts，E.美国，霍普金斯，N. E、Zaluzec，E. J.，Gage，D.一、Alworth，W. L.，和Hollenberg，P. F.（1995）Arch. Biochem. Biophys.323，000-000]。9 EPh的失活导致NADPH支持的7-乙氧基-4-三氟甲基香豆素（EFC）脱乙基反应损失90-95%，但对碘酰苯或异丙苯支持的EFC代谢没有影响。NADPH支持的活性的损失不受细胞色素b5的添加或存在过量的还原酶的影响。在加入甲基非他明后，1型光谱变化的幅度随着9 EPh修饰的蛋白质而降低。无论是用NADPH和还原酶酶促还原还是用连二亚硫酸钠化学还原，修饰的2B 1形成稳定状态水平的CO还原复合物的能力都没有降低，但在厌氧条件下还原酶的还原速率是无底物时天然蛋白的57%，有底物时天然蛋白的35%。与天然2B 1相比，9 EPh修饰的2B 1在苄非他明代谢期间的NADPH氧化、H2 O2形成和甲醛形成速率总体较慢。H2 O2与HCHO的比例为1.0：1.0（天然蛋白）和1.6：1.0（修饰蛋白）。在环己烷存在下，修饰的蛋白质形成稳态水平的氧-铁络合物的能力降低。这些结果是一致的想法，在肽Phe 297中的一个残基的共价修饰Leu 307的菲乙酰基损害还原酶的P450 2B 1的还原，也导致解偶联的NADPH利用和氧消耗的产物形成。
The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization–mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995)Arch. Biochem. Biophys.323, 000–000]. The inactivation by 9EPh resulted in a 90–95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen–iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.