Active site analysis of P450 enzymes: Comparative magnetic circular dichroism spectroscopy
Active site analysis of P450 enzymes: Comparative magnetic circular dichroism spectroscopy
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DOI：
10.1006/abbi.1997.0248
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发表时间：
1997-09-01
影响因子：
3.9
通讯作者：
Peterson, JA
中科院分区：
文献类型：
--
作者：
Andersson, LA;Johnson, AK;Peterson, JA
最近的结构研究表明，P450酶的底物和o -2结合远端口袋并不相同。因此，来自α -萜烯醇代谢假单胞菌的P450terp （CYP108）不同于P450cam (cyp101) (C. A， Hasemann等，J. Mel。生物学。236,1169,1994)。相比之下，P450terp和P450BMP的远端胞袋（CYP102血红素结构域；巨型芽孢杆菌）更为相似，包括远端H2O配体和I螺旋之间的肚脐氢键相互作用（C. A. Hasemann et al., Structure, 3,41 -62, 1995）。为了评估这些差异的意义，我们将P450terp的溶液磁圆二色性（MCD）光谱与其他P450酶的光谱（如P450cam、P450BMP、P450BM-3holo和P450BM1）以及氯过氧化物酶和NO合成酶的光谱进行了比较。与P450cam相比，原生P450terp的光谱与P450BMP和哺乳动物P450LM-2的光谱更接近。在底物结合后，铁P450terp和迄今为止研究的所有其他硫代酸连接血红素系统的MCD光谱显示出一个强大的Soret带，这与过氧化氢酶或其他b -配位铁血红素系统的典型Soret MCD模式相比，明显是独特的。因此，这种强烈的MCD阴性特征可用于诊断半胱氨酸连接铁血红素。在亚铁p450的情况下，sot区MCD槽的强度在底物结合酶和无底物酶之间是不同的（尽管底物不与血红素部分直接接触）。一个特别有趣的新发现是Soret MCD波段在底物结合形式和底物自由形式之间的清晰光谱位移- co - p450 terp。以前没有这样的观察。此外，两种P450terp的波段位置都与已知的ferro - co - p50cam波段发生了红移。因此，这些数据表明，MCD光谱对活性位点极性和H2O占用具有惊人的敏感性，这与远端口袋效应对go结合率和平衡常数的影响一致。将P450terp的光谱特性与其他P450酶的MCD光谱以及与氯过氧化物酶和NO合成酶的光谱特性进行对比分析，发现两者既有预期的相似之处，也有反映活性位点结构特征的显著差异。本文提出的P450terp相对于其他P450酶的详细光谱分析包括首次观察到底物诱导的铁- co -P450光谱移位。此外，本文还对P450-BM-1和新型NO合成酶进行了可测试的结构预测（这两种酶迄今为止都没有结晶）。因此，这项工作提供了对结构定义的P450的见解，也可能导致对其他P450酶的理解。(C) 1997学术出版社。
Recent structural studies indicate that the substrate-and O-2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (CYP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C. A, Hasemann et al., J. Mel. Biol. 236, 1169, 1994). In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain; Bacillus megaterium) are more closely similar, including navel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C. A. Hasemann et al., Structure, 3, 41-62, 1995). To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam. Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other B-coordinate ferric heme systems. This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp. No such observation has been made previously. Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam. These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on GO-binding rates and equilibrium constants. Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450. Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes. (C) 1997 Academic Press.