Enzymatic Δ1-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism
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Other documents of the author: Glanowski, Michał; Wójcik, Patrycja; Procner, Magdalena; Borowski, Tomasz; Lupa, Dawid; Mielczarek, Przemysław; Oszajca, Maria; Świderek, Katarzyna; Moliner, Vicent; Bojarski, Andrzej; Szaleniec, Maciej
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Title
Enzymatic Δ1-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction MechanismAuthor (s)
Date
2021-07-02Publisher
American Chemical SocietyISSN
2155-5435Bibliographic citation
Glanowski, M., Wójcik, P., Procner, M., Borowski, T., Lupa, D., Mielczarek, P., Oszajca, M., Świderek, K.; Moliner, V.; Bojarski, A. J.; Szaleniec, M. Enzymatic Δ1-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism. ACS Catalysis. 2021, 11 (13), 8211-8225, DOI: 10.1021/acscatal.1c01479Type
info:eu-repo/semantics/articlePublisher version
https://pubs.acs.org/doi/abs/10.1021/acscatal.1c01479Version
info:eu-repo/semantics/publishedVersionSubject
Abstract
Δ1-Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ1-KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The ... [+]
Δ1-Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (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 the kinetic isotope effect (KIE) reported in 1960 by Jerussi and Ringold [Biochemistry1965, 4 (10)]. In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4–16.3 kcal·mol–1) and hydride transfer (15.5–18.0 kcal·mol–1) are very close (1.7–2.1 kcal·mol–1), 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. [-]
Is part of
ACS Catalysis, 2021, vol. 11, no 13Funder Name
National Science Centre Poland under the OPUS | Ministerio de Ciencia e Innovación | Generalitat Valenciana | Universitat Jaume I
Project code
UMO-2016/21/B/ST4/03798 | POWR. 03.02.00-00-I013/16 | PGC2018-094852-B-C21 | PID2019-107098RJ-I00 | AICO/2019/195 | SEJI/2020/007 | UJI·B2020-031 | UJI-A2019-04
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info:eu-repo/semantics/openAccess
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