A theoretical model of the catalytic mechanism of the Δ5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr14), aspartate (Asp38) and aspartic acid (Asp99) of the enzyme Δ5-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp38 acts as a base, abstracting the 4β-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6β-position. Each step goes through its own transition state. Functional groups of the Tyr14 and Asp99 side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6–31G* basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.