page_title: Steroid Delta-isomerase
section_title: Mechanism
hierarchical_section_title: Steroid Delta-isomerase / Mechanism
caption_attribution_description: English: Equilenin bound to the ketosteroid isomerase active site from the vantage of the oxyanion hole. (PDB: 1OH0, from pseudomonas putida)
context_page_description: In enzymology, a steroid Δ⁵-isomerase is an enzyme that catalyzes the chemical reaction
  a 3-oxo-Δ⁵-steroid a 3-oxo-Δ⁴-steroid
  Hence, this enzyme has one substrate, a 3-oxo-Δ⁵-steroid, and one product, a 3-oxo-Δ⁴-steroid.
context_section_description: KSI catalyzes the rearrangement of a carbon-carbon double bond in ketosteroids through an enolate intermediate at a diffusion-limited rate. There have been conflicting results on the ionization state of the intermediate, whether it exists as the enolate or enol. Pollack uses a thermodynamic argument to suggest the intermediate exists as the enolate.  The general base Asp-38 abstracts a proton from position 4 (alpha to the carbonyl, next to the double bond) of the steroid ring to form an enolate (the rate-limiting step) that is stabilized by the hydrogen bond donating Tyr-14 and Asp-99. Tyr-14 and Asp-99 are positioned deep within the hydrophobic active site and form a so-called oxanion hole. Protonated Asp-38 then transfers its proton to position 6 of the steroid ring to complete the reaction.
  Although the mechanistic steps of the reaction are not disputed, the contributions of various factors to catalysis such as electrostatics, hydrogen bonding of the oxyanion hole, and distal binding effects are discussed below and still debated.
  The Warshel group applied statistical mechanical computational methods and empirical valence bond theory to previous experimental data.  It was determined that electrostatic preorganization-including ionic residues and fixed dipoles within the active site-contributes most to KSI catalysis. More specifically, Tyr-14 and Asp-99 dipoles work to stabilize the growing charge which accumulates on the enolate oxygen (O-3) throughout catalysis.  In a similar way, the charge on Asp38 is stabilized by surrounding residues and a water molecule during the course of the reaction.  The Boxer group used experimental Stark spectroscopy methods to identify the presence of H-bond-mediated electric fields within the KSI active site.  These measurements quantified the electrostatic contribution to KSI catalysis (70%).
  The active site is lined with hydrophobic residues to accommodate the substrate, but Asp-99 and Tyr-14 are within hydrogen bonding distance of O-3.  The hydrogen bonds from Tyr-14 and Asp-99 are known to significantly affect the rate of catalysis in KSI. Mutagenesis of this residue to alanine (D99A) or asparagine (D99N) results in a loss in activity at pH 7 of 3000-fold and 27-fold, respectively, implicating Asp-99 as important for enzymatic activity. Wu et al. proposed a mechanism that involves both Tyr-14 and Asp-99 forming hydrogen bonds directly to O-3 of the steroid. This mechanism was challenged by Zhao et al., who postulated a hydrogen bonding network with Asp-99 hydrogen bonding to Tyr-14, which in turn forms a hydrogen bond to O-3.  More recently, the Herschlag group utilized unnatural amino acid incorporation to assay the importance of Tyr-14 to KSI catalysis.  The natural tyrosine residue was substituted with unnatural halogenated amino acids surveying a range of pKa's.  There was very little difference in KSI catalytic turnover with decreasing pKa, suggesting, in contrast to the electrostatic studies outlined above, that oxyanion hole stabilization is not primarily important for catalysis.
  Asp-38 general acidic/basic activity and effective molarity was probed by the Herschlag group through site-directed mutagenesis and exogenous base rescue.  Asp-38 was mutated to Gly, nullifying catalytic activity, and exogenous rescue was attempted with carboxylates of varying size and molarity.  By calculating the concentration of base needed for full rescue, the Herschlag group determined the effective molarity of Asp-38 in KSI (6400 M).  Thus, Asp-38 is critical for KSI catalysis.
  Sigala et al. found that solvent exclusion and replacement by the remote hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole.  In addition, ligand binding does not grossly alter the conformations of backbone and side chain groups observed in X-ray structures of PI KSI. However, NMR and UV studies suggest that steroid binding restricts the motions of several active-site groups, including Tyr-16.  Recently, the Herschlag group proposed that remote binding of hydrophobic regi
caption: Close up structure of the KSI (Pseudomonas putida) active site bound to equilenin (aromatic substrate analog) from the vantage point of the oxyanion hole with hydrogen bond lengths (Angstroms) and residue names labeled (PDB: 1OH0).
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