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