Liu Yilin, Denisov Ilia G, Sligar Stephen G, Kincaid James R
Department of Chemistry, Marquette University, 1414W Clybourn Street, Milwaukee, Wisconsin 53233, United States.
J Am Chem Soc. 2021 Mar 17;143(10):3729-3733. doi: 10.1021/jacs.1c00581. Epub 2021 Mar 3.
CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome (Mn-Cyt) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cyt. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.
细胞色素P450 17α-羟化酶1（CYP17A1）是一种重要的人体类固醇生成酶，它催化两个连续反应，分别从孕酮生成雄烯二酮以及从孕烯醇酮生成脱氢表雄酮。第二个反应是C17-C20键断裂，该反应强烈依赖细胞色素的存在，并且当17α-羟基孕酮（17OH-PROG）作为底物时，会表现出一种迄今尚未解释清楚的更为显著的加速作用。细胞色素对CYP17A1催化的C-C键断裂的刺激作用的起源仍存在争议，主要原因要么是电子转移至P450氧复合物的加速，要么是细胞色素的变构效应有利于促进裂解酶活性的活性位点构象。我们使用共振拉曼光谱，比较了锰取代的细胞色素（Mn-Cyt）对CYP17A1氧复合物的影响，这两种蛋白质都共掺入脂质纳米盘。对于含有17OH-PROG的CYP17A1，在存在锰时观察到Fe-O模式的特征性位移，表明底物的17OH基团与Fe-O-O部分的末端氧原子之间的氢键重新定向为与近端氧原子形成氢键，这种构型有利于裂解酶催化。对于17α-羟基孕烯醇酮，未观察到这种位移，即使没有Mn-Cyt，也存在有利的氢键取向。这些新数据为17OH-PROG底物更显著的加速作用提供了精确的变构解释。
