In enzymology, a steroid Δ5-isomerase (EC 5.3.3.1) is an enzyme that catalyzes the chemical reaction
a 3-oxo-Δ5-steroid ⇌ {\displaystyle \rightleftharpoons } a 3-oxo-Δ4-steroid Hence, this enzyme has one substrate, a 3-oxo-Δ5-steroid, and one product, a 3-oxo-Δ4-steroid.
Introduction This enzyme belongs to the family of isomerases, specifically those intramolecular oxidoreductases transposing C=C bonds. The systematic name of this enzyme class is 3-oxosteroid Δ5-Δ4-isomerase. Other names in common use include ketosteroid isomerase (KSI), hydroxysteroid isomerase, steroid isomerase, Δ5-ketosteroid isomerase, Δ5(or Δ4)-3-keto steroid isomerase, Δ5-steroid isomerase, 3-oxosteroid isomerase, Δ5-3-keto steroid isomerase, and Δ5-3-oxosteroid isomerase. KSI has been studied extensively from the bacteria Comamonas testosteroni (TI), formerly referred to as Pseudomonas testosteroni, and Pseudomonas putida (PI). The enzymes from these two sources are 34% homologous, and structural studies have shown that the placement of the catalytic groups in the active sites is virtually identical. Mammalian KSI has been studied from bovine adrenal cortex and rat liver. This enzyme participates in c21-steroid hormone metabolism and androgen and estrogen metabolism. An example substrate is Δ5-androstene-3,17-dione, which KSI converts to Δ4-androstene-3,17-dione. The above reaction in the absence of enzyme takes 7 weeks to complete in aqueous solution. KSI performs this reaction on an order of 1011 times faster, ranking it among the most proficient enzymes known. Bacterial KSI also serves as a model protein for studying enzyme catalysis and protein folding.
Structural studies KSI exists as a homodimer with two identical halves. The interface between the two monomers is narrow and well defined, consisting of neutral or apolar amino acids, suggesting the hydrophobic interaction is important for dimerization. Results show that the dimerization is essential to function. The active site is highly apolar and folds around the substrate in a manner similar to other enzymes with hydrophobic substrates, suggesting this fold is characteristic for binding hydrophobic substrates. No complete atomic structure of KSI appeared until 1997, when an NMR structure of TI KSI was reported. This structure showed that the active site is a deep hydrophobic pit with Asp-38 and Tyr-14 located at the bottom of this pit. The structure is thus entirely consistent with the proposed mechanistic roles of Asp-38 and Tyr-14.
As of late 2007, 25 structures have been solved for this class of enzymes, with PDB accession codes PDB: 1BUQ, PDB: 1C7H, PDB: 1CQS, PDB: 1DMM, PDB: 1DMN, PDB: 1DMQ, PDB: 1E97, PDB: 1GS3, PDB: 1ISK, PDB: 1K41, PDB: 1OCV, PDB: 1OGX, PDB: 1OGZ, PDB: 1OH0, PDB: 1OHO, PDB: 1OHP, PDB: 1OHS, PDB: 1OPY, PDB: 1VZZ, PDB: 1W00, PDB: 1W01, PDB: 1W02, PDB: 1W6Y, PDB: 2PZV, and PDB: 8CHO.
Mechanism
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.
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