Collom Samuel L, Jamakhandi Arvind P, Tackett Alan J, Radominska-Pandya Anna, Miller Grover P
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, AR 72205, USA.
Arch Biochem Biophys. 2007 Mar 1;459(1):59-69. doi: 10.1016/j.abb.2006.10.028. Epub 2006 Nov 2.
Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.
尽管其具有生物学重要性，但我们对细胞色素P450 2E1（CYP2E1）催化过程中活性位点结构及关键氨基酸的相关性了解仍然有限。在本研究中，我们通过用7-叠氮基-4-甲基香豆素（AzMC）进行光亲和标记并结合CYP2E1同源模型来鉴定CYP2E1活性位点残基。在无光条件下，AzMC是CYP2E1催化底物对硝基苯酚氧化的有效竞争性抑制剂。AzMC的光活化导致CYP2E1活性和结构完整性呈浓度依赖性丧失，这是由于血红素和蛋白质均发生了修饰。光标记反应使血红素降解并产生了一种可能的血红素加合物。探针掺入蛋白质发生在底物识别序列5（SRS-5）内的多个位点。基于CYP2E1同源模型，我们推测AzMC标记了活性位点中的SRS-5残基Leu363、Val364和Leu368。此外，我们提出一系列苯丙氨酸，尤其是Phe106，介导与香豆素的相互作用。
