Solution Structure of 3-Oxo-Δ 5 -Steroid Isomerase

Abstract

The three-dimensional structure of the enzyme 3-oxo-Δ 5 -steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three α helices and a six-strand mixed β-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr 14 (general acid) and Asp 38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp 99 ) is also located in the active site adjacent to Tyr 14 , and kinetic and binding studies of the Asp 99 to Ala mutant demonstrate that Asp 99 contributes to catalysis by stabilizing the intermediate.