Studies on the characterization of human sterol 8-isomerase and biosynthesis of sterols in Pneumocystis carinii

Date

2002-08

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Publisher

Texas Tech University

Abstract

Two studies were performed to gain insight into sterol biosynthesis in humans and in an opportunistic fungus which can generate mortality for AIDS patients.

In Study 1, the cDNA {CHOI), encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg2 cells and purified to apparent homogeneity. The enzyme exhibited aKm of 50 µM and a turnover number of 0.423 s ^(-1) for zymosterol, an isoelectric point of 7.70, a native molecular weight 107,000 Da and was tetrameric. From a series of incubations with substrate mimics of zymosterol, the equatorial geometry and hydrogen bonding ability of the C-3 hydroxyl group and native length of the side chain of zymosterol were found to be obligatory for substrate acceptability. Biomimetic studies of acid-catalyzed isomerization of zymosterol resulted in formation of cholest-8(14)-sterol, whereas the enzyme generated product was a Ä^7-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using 2H20 and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. Kinetic analyses indicated the reaction equilibrium (Keq = 14; ÄG°'= -6.5 kJ/mol) for isomerization favored the forward direction leading to Ä^7 sterol formation. Treatment of hSI with different high energy intermediate analogs assayed against zymosterol produced the following dissociation constants: emopamil (Ki 2 µM) = tamoxifen (Ki 1 µM) = tridemorph {Ki 1 µM) < 25-azacholesterol (Ki 21 µ,M) < ketoconazole (Ki 156 µM) < cholesterol (Ki 620 µ.M). Dissociation constants for a set of emopamil analogues prepared by chemists at AstraZeneca Co. were also determined. M373931 was the most highly potent drug exhibiting a Ki of 50 nM and a competitive type kinetic pattern.

hSI contains amino acid side chains at E80, S133 and R147 which are sensitive to missense mutation. To evaluate the roles of these amino acids, a series of mutants were prepared from the parent hSI hybrid and BL21(DE3) cells. E80 was replaced with K or A, SI33 was replaced with R, Y or A, and R147 was replaced with K or A. The kinetic parameters of zymosterol and emopamil generated from the mutant hSI enzymes indicate the amino acids are important to the proper conformation of the active center contributing to catalysis.

In Study 2, the sterol composition of P. carinii was analyzed. Thirty-three sterols were detected in the organism. The structures of the compounds were determined by chromatographic and spectral techniques. Cholesterol was found to be the major cellular sterol (80% of total sterols). 24-Alkyl sterols composed the bulk of the remaining cellular sterols (18% of total sterols). Based on the sterol composition of the cell, a novel biosynthetic pathway is proposed in which sterol methyl fransferase serves as the ratelimiting enzyme in phytosterol synthesis.

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