Solution Structure of 3-Oxo-$\Delta^5$-Steroid Isomerase

• Published in: Science (American Association for the Advancement of Science) 1997-04, Vol.276 (5311), p.415-418
• Main Authors: Wu, Zheng Rong, Ebrahimian, Soheila, Zawrotny, Michael E., Thornburg, Lora D., Perez-Alvarado, Gabriela C., Brothers, Paul, Pollack, Ralph M., Summers, Michael F.
• Format: Article
• Language: English
• Subjects: Active sites; Atoms; Biochemistry; Dimers; Enzymes; Hydrogen bonds; Kinetics; Monomers; Nuclear magnetic resonance; Protons
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.276.5311.415

The three-dimensional structure of the enzyme 3-oxo-$\Delta^5$-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.