BILE SALT-MODULATED STEREOSELECTION IN THE CHOLESTEROL ESTERASE-CATALYZED HYDROLYSIS OF ALPHA-TOCOPHERYL ACETATES

The noncompetitive and-competitive hydrolyses of (2R, 4'R, 8'R)-alpha- tocopheryl acetate (RRR-alpha-TOAc, the acetate of natural vitamin E) and (2S,4'R,8'R)-alpha-tocopheryl acetate (SRR-alpha-TOAc) catalyzed b y crude and pure bovine cholesterol esterase (BCE) and crude and pure porcine cholesterol esterase (PCE) have been studied at 37 degrees C. These two CE's are catalytically active toward tocopheryl acetates onl y in the presence of bile salts. The three 3 alpha,7 alpha,12 alpha-tr ihydroxy bile salts, cholate, glycocholate and taurocholate, not only are effective activators of BCE and PCE, but also modulate the diaster eoselectivities of these two enzymes in their hydrolyses of RRR- and S RR-alpha-TOAc in a characteristic manner. Rates of hydrolyses were muc h faster in the presence of a small quantity of dl- or l-dimyristoylph osphatidylcholine (DMPC) than in its absence. However, for each enzyme , the direction and even the magnitude of the diastereoselectivity is primarily determined by the bile salt employed and not by the presence or nature of the co-lipid (DMPC or sodium oleate), nor by the bile sa lt/co-lipid ratio, nor by the purity of the enzyme. In noncompetitive experiments, the ratios of the BCE-catalyzed initial rates of hydrolys es of the diastereomeric acetates, V-i(RRR)/V-i(SRR), are 0.21, 1.5, a nd 2.7 for cholate, glycocholate, and taurocholate, respectively, and for the PCE-catalyzed noncompetitive reactions, 0.21, 7,9, and 7.5 for the same three bile salts. In competitive experiments using equal con centrations of RRR- and SRR-alpha-TOAc, the BCE-catalyzed initial rate ratios are 0.33, 0.94, and 2.2, and for the PCE-catalyzed competitive reactions, 0.21, 1.1, and 1.8 for cholate, glycocholate, and taurocho late, respectively. The lower diastereoselectivities found in some of the competitive experiments are tentatively attributed to competitive inhibition of the enzyme by one of the diastereomeric acetates or, mor e probably, its phenolic product. Glycochenodeoxycholate, a 3 alpha,7 alpha-dihydroxy bile salt, activates PCE but not BCE. With the former enzyme, the diastereoselectivities, V-i(RRR)/V-i(SRR) are rather simil ar for the noncompetitive and competitive experiments, viz., 11 and 8. 0, respectively, and the reaction rates are comparable to those found with the trihydroxy bile salts. Since the detailed composition of the bile salt/co-lipid mixed micelle does not determine V-i(RRR)/V-i(SRR) ratios, we conclude that diastereoselectivities are not determined by the precise surface structure of the micelle. Presumably, the bile sal ts modulate the diastereoselectivities of BCE and PCE by a direct effe ct on the protein which may involve ''refolding'' the enzyme with a co nsequent change in the shape of the active site. It should be noted th at the relevant chiral carbon in alpha-TOAc is separated by six bonds from the bond which is broken and that this chiral center must exert i ts influence prior to the rate-limiting step in the overall hydrolysis . If the bile salt modulating effect on CE-catalyzed reactions is to b e exploited in organic syntheses, it is the competitive experiments wh ich are the more important, and for these, the diastereoselectivity, V -i(RRR)/V-i(SRR), varies from a low of 0.21 for the cholate/PCE couple to a high of 8.0 for the glycochenodeoxycholate/PCE couple. Such a dr amatic 40-fold change in an enzyme's chiral selectivity is without pre cedent.