Cvrk T, Strobel H W
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston 77225, USA.
Arch Biochem Biophys. 1998 Jan 1;349(1):95-104. doi: 10.1006/abbi.1997.0464.
Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to beta-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the cumene hydroperoxide binding region in the cytochrome P450 active site.
在缺乏分子氧、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和细胞色素P450 - NADPH氧化还原酶的情况下，氢过氧化异丙苯可以支持细胞色素P450催化的反应。其在细胞色素P450活性位点的结合受氢过氧化异丙苯结合区域结构的支配。为了确定细胞色素P4501A1上氢过氧化异丙苯结合的区域，我们制备了一种氢过氧化异丙苯类似物用作光亲和标记物。对叠氮对异丙基苯（叠氮异丙苯）及其氚化衍生物在水溶液中用紫外光进行光解，半衰期为29秒。在氢过氧化异丙苯支持的体系中，由P450催化的7 - 乙氧基香豆素脱乙基反应受到该标记物的强烈抑制。在氢过氧化异丙苯存在的情况下，光活化后与蛋白质的共价结合被阻断了50%。对标记蛋白质进行胰蛋白酶消化后的高效液相色谱（HPLC）分析表明，[3H] - 叠氮异丙苯共价连接到与1A1序列中492 - 503位残基相对应的肽VDMTPAYGLTLK上。当在氢过氧化异丙苯存在下进行标记时，该部分的放射性水平降低了50%。为了确认所确定的区域，用溴化氰裂解标记的蛋白质。对溴化氰消化产物进行HPLC分离显示出两个具有高放射性水平的峰。对洗脱时间为43分钟的放射性部分进行十二烷基硫酸钠/三羟甲基氨基甲烷聚丙烯酰胺凝胶电泳（SDS/Tricine PAGE）分析，发现一个2.4 kDa的肽带有高水平的共价结合放射性。N端序列确定该标记肽是由溴化氰产生的对应于494 - 512位残基的片段。洗脱时间为27分钟的标记肽的N端序列TLKH与细胞色素P4501A1序列中的501 - 504位氨基酸残基匹配。我们可以得出结论，在所有三个确定的肽的重叠区域T501 - L502 - K503是叠氮异丙苯与细胞色素P4501A1共价结合的位点。细胞色素P4501A1与细胞色素P450102的序列比对预测，该区域可能对应于位于血红素环远侧靠近I螺旋和氧结合口袋的β - 折叠结构。据我们所知，这是首次报道在细胞色素P450活性位点定位氢过氧化异丙苯结合区域。
