Enzymatic Δ1-dehydrogenation of 3-ketosteroids – Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Data and analysis supporting the publication titled 'Enzymatic Δ1-dehydrogenation of 3-ketosteroids – Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism' (2021) created by Michał Glanowski, Patrycja Wójcik, Magdalena Procner, Tomasz Borowski, Dawid Lupa, Przemysław Mielczarek, Maria Oszajca, Katarzyna Świderek, Vicent Moliner, Andrzej J. Bojarski, Maciej Szaleniec, ACS Catalysis 2021, 11, 8211−8225. Online access: https://doi.org/10.1021/acscatal.1c01479 Δ1-Dehydrogenation of 3-ketosteroids catalyzed by FAD-dependent 3-ketosteroid dehydrogenases (Δ1-KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The catalytic mechanism assumes the formation of a double bond in two steps, proton abstraction by tyrosyl ion and a rate-limiting hydride transfer to FAD. This hypothesis was never verified by quantum-mechanical studies despite contradictory results from kinetic isotope effect (KIE) reported in ’60 by Jerussi and Ringold (Biochemistry 1965, 4 (10)). In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4-16.3 kcal/mol) and hydride transfer (15.5-18.0 kcal/mol) are very close (1.7-2.1 kcal/mol) which explains normal KIE values for steroids labeled either at C1 or C2 atoms. We confirm that tyrosyl ion acting as the catalytic base is indeed necessary for efficient activation of the steroid. We explain the lower value of the observed KIE (1.5-3.5) by the nature of the free energy surface, the presence of diffusion limitation and to a smaller extent conformational changes of the enzyme upon substrate binding. Finally, we confirm the Ping-Pong bi bi kinetics of the whole Δ1-dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation and enzyme reoxidation proceed at comparable rates. This repository contains data acquired in this study i.e., raw data from stopped-flow spectrophotometer used to obtain kinetic traces for steady-state and pre-steady-state kinetics, including measurements of the kinetic isotope effect. The data were fitted with kinetic models yielding kinetic constants and confirming the Ping-Pong bi bi mechanism. The pre-steady-state kinetics conducted at different micro and macroviscosites were used to measure Kinetic Solvent Viscosity Effects (KSVE). Furthermore, a pre-steady-state experiment with 17-methyltestosterone was subjected to a global-fitting procedure in Octave which resulted in establishing microkinetic constants of substrate binding and release, constant of substrate oxidation/FAD reduction as well as of the reverse process. The authors acknowledge financial support from the National Science Centre Poland under the OPUS grant number UMO-2016/21/B/ST4/03798.本数据集包含支撑题为《3-酮类固醇的酶促Δ¹脱氢——动力学同位素效应与反应机制的统一》的论文发表所需的数据与分析，该论文于2021年发表于《ACS Catalysis》（2021, 11, 8211−8225），作者为Michał Glanowski、Patrycja Wójcik、Magdalena Procner、Tomasz Borowski、Dawid Lupa、Przemysław Mielczarek、Maria Oszajca、Katarzyna Świderek、Vicent Moliner、Andrzej J. Bojarski、Maciej Szaleniec。在线访问链接：https://doi.org/10.1021/acscatal.1c01479 由黄素腺嘌呤二核苷酸（Flavin Adenine Dinucleotide, FAD）依赖性3-酮类固醇脱氢酶（Δ¹-KSTD）催化的3-酮类固醇Δ¹脱氢反应，是类固醇降解与多种类固醇药物合成的关键步骤。该催化机制假设双键的形成分为两步：酪氨酸离子夺取质子，以及限速步骤的氢化物向FAD转移。尽管1960年代Jerussi与Ringold（发表于《Biochemistry》1965, 4(10)）报道的动力学同位素效应（Kinetic Isotope Effect, KIE）结果存在矛盾，但该机制假说从未通过量子力学研究得到验证。本研究中，我们展示了可使机制假说与实验证据相统一的结果。量子力学/分子力学分子动力学（Quantum Mechanics/Molecular Mechanics Molecular Dynamics, QM/MM MD）模拟表明，所提出的机制确实是最可能的路径，但与底物活化（13.4~16.3 kcal/mol）和氢化物转移（15.5~18.0 kcal/mol）相关的能垒非常接近（1.7~2.1 kcal/mol），这解释了在C1或C2原子处标记的类固醇所呈现的正常KIE值。我们证实了作为催化碱的酪氨酸离子确实是类固醇高效活化所必需的。我们通过自由能面的性质、扩散限制的存在以及较小程度上底物结合时酶的构象变化，解释了观测到的KIE值较低（1.5~3.5）的原因。最后，我们验证了整个Δ¹脱氢反应的乒乓双底物双产物（Ping-Pong bi bi）动力学，并证明底物结合、类固醇脱氢与酶再氧化以相近速率进行。 本仓库包含本研究中获取的全部数据，即用于获取稳态与预稳态动力学轨迹的停流分光光度计原始数据，包括动力学同位素效应的测量数据。这些数据通过动力学模型拟合得到了动力学常数，并验证了乒乓双底物双产物机制。在不同微观与宏观黏度下开展的预稳态动力学实验被用于测定动力学溶剂黏度效应（Kinetic Solvent Viscosity Effects, KSVE）。此外，针对17-甲基睾酮的预稳态实验在Octave中完成了全局拟合，由此确定了底物结合与释放的微观动力学常数、底物氧化/FAD还原的常数以及逆反应常数。 作者感谢波兰国家科学中心通过OPUS项目（编号UMO-2016/21/B/ST4/03798）提供的经费支持。