Cupp-Vickery J, Anderson R, Hatziris Z
Department of Chemistry and Biochemistry, California State University, 800 North State College Boulevard, Fullerton, CA 92834, USA.
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3050-5. doi: 10.1073/pnas.97.7.3050.
Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify structural factors contributing to steroid and polycyclic aromatic hydrocarbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P450eryF from Saccharopolyspora erythraea with androstenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P450eryF binds androstenedione with an affinity of 365 microM and a Hill coefficient of 1.31 +/- 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 microM and a Hill coefficient of 1.38 +/- 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P450eryF were grown and diffracted to 2.1 A and 2.35 A, respectively. Electron density maps indicate that for both complexes two ligand molecules are simultaneously present in the active site. The P450eryF/androstenedione model was refined to an r = 18.9%, and the two androstenedione molecules have similar conformations. The proximal androstenedione is positioned such that the alpha-face of carbon-6 is closest to the heme iron, and the second steroid molecule is positioned 5.5 A distal in the active site. The P450eryF/9-aminophenanthrene model was refined to an r = 19.7% with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned approximately 6 A distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major conformational changes in the protein.
几种哺乳动物细胞色素P450（P450）同工型与许多底物表现出同促协同作用，这些底物包括类固醇和多环芳烃。为了确定有助于类固醇和多环芳烃与P450酶结合的结构因素，并确定变构位点的位置，我们研究了来自糖多孢红霉菌的大环内酯羟化酶P450eryF与雄烯二酮和9-氨基菲的相互作用。光谱结合分析表明，P450eryF与雄烯二酮的结合亲和力为365μM，希尔系数为1.31±0.6，与9-氨基菲的配位亲和力为91μM，希尔系数为1.38±0.2。生长了雄烯二酮和9-氨基菲与P450eryF的复合物晶体，其衍射分辨率分别为2.1 Å和2.35 Å。电子密度图表明，对于这两种复合物，活性位点中同时存在两个配体分子。P450eryF/雄烯二酮模型精修至r = 18.9%，两个雄烯二酮分子具有相似的构象。近端雄烯二酮的位置使得碳-6的α面最接近血红素铁，第二个类固醇分子位于活性位点中距离其5.5 Å的远端。P450eryF/9-氨基菲模型精修至r = 19.7%，近端9-氨基菲通过9-氨基与血红素铁配位，第二个配体位于活性位点中距离其约6 Å的远端。这些结果表明，配体结合中的同促协同作用可能源于活性位点口袋内两个底物分子的结合，而蛋白质没有发生重大构象变化。
