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

Abstract

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 Km of 50μM and a turnover number of 0.423s-1 for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Δ7-sterol, 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 cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium (Keq = 14; ΔGo′ = −6.5kJ/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 (Ki): emopamil (2μM) = tamoxifen (1μM) = tridemorph (1μM)<25-azacholesterol (21μM) <ketoconazole (156μM)<cholesterol (620μM). The results were consistent with stereoelectronic features of isomerization and support the general model for Δ7-sterol formation in cholesterol synthesis.