Crystal Structure of Δ5-3-Ketosteroid Isomerase from Pseudomonas testosteroni in Complex with Equilenin Settles the Correct Hydrogen Bonding Scheme for Transition State Stabilization

• Published in: The Journal of biological chemistry 1999-11, Vol.274 (46), p.32863-32868
• Main Authors: Cho, Hyun-Soo, Ha, Nam-Chul, Choi, Gildon, Kim, Hyun-Ju, Lee, Donghan, Oh, Kyung Seok, Kim, Kwang S., Lee, Weontae, Choi, Kwan Yong, Oh, Byung-Ha
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.46.32863

Δ5-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp38 (pKa ∼4.7) was identified as the general base abstracting the steroid C4β proton (pKa ∼12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 Å crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr14 OH and Asp99 COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp99 donates the hydrogen bond to Tyr14, which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr14 OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.