SOLUTION STRUCTURE OF 3-OXO-DELTA(5)-STEROID ISOMERASE

The three-dimensional structure of the enzyme 3-oxo-Delta(5)-steroid i somerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. Th e two independently folded monomers pack together by means of extensiv e hydrophobic and electrostatic interactions. Each monomer comprises t hree alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Ty r(14) (general acid) and Asp(38) (general base) are located near the b ottom of the cavity and positioned as expected from mechanistic hypoth eses. 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.