Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Abstract

Δ 1 -Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ 1 -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 [Biochemistry 1965, 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 Δ 1 -dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation, and enzyme reoxidation proceed at comparable rates.

Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new membrane-associated domain and proton-relay system in catalysis

“…The modeling revealed that the reaction mechanism catalyzed by AcmB proceeds according to the classical mechanism postulated in the literature and recently confirmed by our calculations for KstD1 from R. erythropolis 26 (Figure 5). The enzyme binds a steroid substrate (E:S), so its ring A is positioned almost parallel to the isoalloxazine ring system of FAD.…”

“…Kinetic experiments have confirmed that the reaction catalyzed by AcmB proceeds according to Ping-Pong bi-bi mechanisms, as recently reported by us for KstD1 from R. erythropolis 26 . AcmB exhibited a high affinity for progesterone with KmA value of 4.4 ± 0.3 μM and a 20-fold lower affinity for DCPIP (KmB 79.3 ± 6.9 μM).…”

“…The details on the procedure are available in SI. The FAD content in the expressed enzymes was estimated as the ratio of the spectrophotometrically measured AcmB-FAD concentration (εAcmB 450 nm = 12 094 M -1 cm -1 , determined as in 26 ) to the total protein concentration determined according to the Bradford method 27 .…”

“…The steady-state kinetic studies leading to establishing the kinetic mechanism were conducted with stopped-flow according to the previously described methodology 26 .…”

“…The QM layer consisted of the steroid substrate, Y363, and the FAD fragment (similarly to our last study 26 ), while other residues were treated with the AMBER forcefield as implemented in fDynamo library 41,42 . The positions of residues beyond 20 Å from the substrate were fixed.…”