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.
Steps to reproduce
The GLOBAL_FITTING_OCTAVE directory contains octave scripts for global kinetic adjustment. All inputs are included as well as the results. To run a global match, open and run the pre_eq_v01_4_D.m script. In the file STEADY-STATE KINETIC EXPERIMENTS we present raw data to obtain kinetic traces followed at 700 nm for reaction mixture contained Tris-HCl buffer pH 8.0, KSTD, varying concentration of 2,6-dichloroindophenol (DCPIP) and AD in isopropanol. The kinetic measurements were repeated at least 5 times for each AD concentration. The initial rate constants were obtained by fitting a linear function to the first 10 seconds of the studied reaction. Subsequently, received data were used to fit the non-sequential Ping-Pong bi bi model with non-linear regression. The file 17-MT KINETIC presents raw data for pre-steady state kinetic of FAD reduction in the reaction of KSTD with the 17-MT using Tris-HCl pH 8.0. The obtained traces (8 repetitions for each 17-MT concentration) were followed at 450 nm for 31.25 ms and subsequently were globally fitted to a kinetic model (a set of differential equations) consisting of two consecutive reversible steps: formation/dissociation of ES complex (k1 and k-1) and FAD reduction/oxidation by the substrate/product (k2 and k-2): ES = E’P. The raw data showed in this file allowed us to determine KD and kcat values for the discussed reaction. Files KSVE-GLYCEROL and KSVE-PEG present raw data for the kinetic solvent viscosity effect (KSVE), which was determined by pre-steady-state kinetics measurements for the reaction mixture contained Tris-HCl buffer pH 8.0, KSTD, varying concentration of AD in 2-methoxyethanol (EGME) and glycerol or PEG 20 000. The kinetic traces were followed at 450 nm for 35 to 125 ms. Obtained traces were fitted with single exponential functions. Subsequently, collected data were fitted to the Michaelis – Menten model with non-linear regression. Files KIE C1 and KIE C2 present data for the kinetic isotope effect at C1 and C2 atoms of the 3-ketosteroid core. The data were determined by a direct method under steady-state or pre-steady-state conditions. The reaction velocities were determined in a spectrophotometric activity assay. The measurements were carried out in Tris-HCl buffer pH 8.0 with DCPIP, steroid dissolved in dioxane and KSTD. The reduction of DCPIP was followed at 700 nm. All measurements were performed in triplicates. The initial rate constants were obtained with linear regression fitted to the initial parts of the kinetic curves. The observed rate constant for C1 was determined in the reaction of KSTD with either DHT or 1,16,16,17-d4-DHT in EGME/buffer solution. The observed rate constant for the C2 atom was measured with KSTD and either 17-MT or 2,2,4,6,6-d5-17-MT analog dissolved in dioxane/buffer solution. Each experiment was repeated at least 12 times. The obtained traces were fitted with single exponential functions and the ratio of the average kobsH/kobsD was determined.