Mechanism
KSI catalyzes a C-H bond cleavage and formation through an enolate intermediate at a diffusion-limited rate. The general base Asp-38 abstracts a proton from position 4 of the steroid ring to form an enolate that is stabilized by the hydrogen bond donating Tyr-14 and Asp-99. Tyr-14 and Asp-99 are positioned deep within the hydro-phobic 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. The hydrogen bonds from Tyr-14 and Asp-99 are known to significantly affect the rate of catalysis in KSI.
The active site pit is lined with hydrophobic residues, but there exists an ionic residue, Asp-99, located adjacent to Tyr-14 and within hydrogen bonding distance of O-3. 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.
Numerous physical changes occur upon steroid binding within the KSI active site. In the free enzyme an ordered water molecule is positioned within hydrogen-bonding distance of Tyr-16 (the PI equivalent of TI KSI Tyr-14) and Asp-103 (the PI equivalent of TI KSI Asp-99). This and additional disordered water molecules present within the unliganded active site are displaced upon steroid binding and are substantially excluded by the dense constellation of hydrophobic residues that pack around the bound, hydrophobic steroid skeleton. 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.
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.
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.
Read more about this topic: Steroid Delta-isomerase
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