From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
J Biol Chem. 2019 Jun 28;294(26):10028-10041. doi: 10.1074/jbc.RA119.008860. Epub 2019 May 9.
Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.
细胞色素 P450（P450，CYP）酶是参与类固醇以及许多其他化合物氧化的主要催化剂。其多功能性部分可以通过蛋白质的灵活性和结合机制的复杂性来解释。然而，这些蛋白质是否通过诱导契合或构象选择来结合其底物尚不清楚。P450 17A1 在类固醇生成中起主要作用，通过 17α-羟基（OH）中间体分别催化孕酮和孕烯醇酮的两步氧化，生成雄烯二酮和脱氢表雄酮。我们通过分析进展曲线（紫外可见光谱）研究了 P450 17A1 与类固醇底物的相互作用，结果表明，这些底物中任何一种的结合速率都随底物浓度的增加而降低，这是构象选择的标志。此外，当 17α-OH 孕烯醇酮的浓度保持不变而 P450 浓度增加时，结合速率增加，这种相反的模式也可诊断为构象选择。动力学模拟建模也更符合构象选择，而不是诱导契合机制。细胞色素 P450 部分通过改变 P450 17A1 的构象来增强 P450 17A1 裂解酶的活性，但在判断 17α-OH 孕烯醇酮或 17α-OH 孕酮的结合时，并没有明显改变它们的结合，这是由表观和结合动力学决定的。P450 17A1 抑制剂阿比特龙也以多步方式与 P450 17A1 结合，建模表明，药物orteronel 对 P450 17A1 的两步选择性抑制只能通过多构象模型来合理化。总之，P450 17A1 通过构象选择结合其类固醇底物。
