Department of Chemistry, Kalamazoo College, Kalamazoo, Michigan.
Department of Chemistry, Kalamazoo College, Kalamazoo, Michigan
Drug Metab Dispos. 2021 Mar;49(3):179-187. doi: 10.1124/dmd.120.000274. Epub 2020 Dec 29.
CYP2D6 is a major drug metabolizing enzyme with a buried active site. Channels leading to the active site from various enzyme surfaces are believed to facilitate ligand egress and access to the active site. The present study used molecular dynamics (MD) and in vitro studies with CYP2D61 and a Trp75-to-Ala mutant to examine channel gating in CYP2D6 by Trp75. MD simulations measured energy landscapes of Trp75 conformations and simulated substrate passage within channel 2b using bufuralol as a model substrate. Trp75 alternated between multiple stable states that supported substrate transport along channel 2b with low-energy barriers between states (∼ -1 kcal/mol). Trp75 conformations were stabilized primarily by hydrogen bonding between Trp75 and Glu222, Asn226, Ala225, or Gln72. Energy barriers were low between Trp75 conformations, allowing Trp75 to easily move between various conformations over time and to function in both binding to and moving substrates in the 2b channel of CYP2D6. Michaelis-Menten kinetic studies completed with purified enzyme in a reconstituted system showed overall reduced enzyme efficiency for metabolism of bufuralol and dextromethorphan by the Trp75Ala mutant compared with CYP2D61. In stopped-flow measurements, for dextromethorphan was decreased in the absence of Trp75. Our results support a role for Trp75 in substrate shuttling to the active site of CYP2D6. SIGNIFICANCE STATEMENT: Using combined molecular dynamics and in vitro assays, this study shows for the first time a role for Trp75 as a channel entrance gating residue in the mechanism of substrate binding/unbinding in CYP2D6. Energy landscapes derived from molecular dynamics were used to quantitate the strength of gating, and kinetics assays showed the impact on enzyme efficiency and of a Trp75Ala mutation.
CYP2D6 是一种主要的药物代谢酶，具有一个埋藏的活性位点。人们认为，通向各种酶表面的活性位点的通道有助于配体的逸出和进入活性位点。本研究使用分子动力学 (MD) 和 CYP2D61 与色氨酸 75 到丙氨酸突变体的体外研究，检查色氨酸 75 在 CYP2D6 中的通道门控作用。MD 模拟测量了色氨酸 75 构象的能量景观，并使用布夫洛尔作为模型底物模拟了通道 2b 内的底物通过。色氨酸 75 在多个稳定状态之间交替，这些状态支持沿着通道 2b 的底物运输，状态之间的能量壁垒较低（约-1 千卡/摩尔）。色氨酸 75 的构象主要通过色氨酸 75 与 Glu222、Asn226、Ala225 或 Gln72 之间的氢键稳定。色氨酸 75 构象之间的能量壁垒较低，允许色氨酸 75 随时间在各种构象之间轻松移动，并在 CYP2D6 的 2b 通道中结合和移动底物的功能。在重建系统中用纯化酶完成的米氏动力学研究表明，与 CYP2D61 相比，色氨酸 75 突变为丙氨酸的突变体对布夫洛尔和右美沙芬代谢的总体酶效率降低。在停流测量中，在没有色氨酸 75 的情况下，右美沙芬的 减少。我们的结果支持色氨酸 75 在底物转运到 CYP2D6 活性位点中的作用。意义声明：本研究首次使用组合分子动力学和体外测定法，证明色氨酸 75 作为通道入口门控残基在 CYP2D6 中底物结合/解吸机制中的作用。从分子动力学得出的能量景观用于定量门控的强度，而动力学测定表明了色氨酸 75 突变为丙氨酸对酶效率和 的影响。
