Purification, characterization and catalytic properties of human sterol 8-isomerase

CHO2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K_m of 50 μM and a turnover number of 0.423 s^-1 for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest8(14)-enol, whereas the enzyme-generated product was a Δ⁷sterol, suggesting absolute stereochemical control of the reaction by hSI. Using ²H₂O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9α was established by a combination of ¹H- and ¹³C-NMR analyses of the enzyme-generated cholesta7,24-dienol. Kinetic analyses indicated the reaction equilibrium (K_eq = 14; ΔG^o′ = - 6.5 kJ/mol) for double-bond isomerization favoured the forward direction, δ⁸ to Δ⁷. Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants (K_i): emopamil (2 μM) = tamoxifen (1 μM) = tridemorph (1 μM) < 25-azacholesterol (21 μM) < ketoconazole (156 μM) < cholesterol (620 μM). The results were consistent with stereoelectronic features of isomerization and support the general model for Δ⁷-sterol formation in cholesterol synthesis.