Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of beta, gamma-unsaturated 3-oxosteroids to their conjugated isomers through the for mation of an intermediate dienolate. The three-dimensional structure of the enzyme from Pseudomonas testosteroni was solved by multidimensional hetero nuclear magnetic resonance spectroscopy. This protein, a 28-kDa symmetric d imer, exhibits a three-dimensional fold with the two independently folded m onomers packed together via extensive hydrophobic and electrostatic interac tions. The previously identified catalytically important residues Tyr-14 (g eneral acid) and Asp-38 (general base) are located near the bottom of a dee p hydrophobic cavity and are positioned in a manner consistent with previou s mechanistic hypotheses. The structure also revealed the presence of an un expected acid group (Asp-99) located in the active site adjacent to Tyr-14. Mutagenesis and kinetic studies show that Asp-99 has an anomalously high p K(a) (>9), which allows it to contribute to catalysis by donating a hydroge n bond to the intermediate and to the transition states. In support of this hypothesis, effects on the kinetic parameters of the mutations Y14F and D9 9A are additive in the Y14F/D99A mutant. (C) 1999 Academic Press.