Bile salt-modulated stereoselection in the cholesterol esterase-catalyzed hydrolysis of α-tocopheryl acetates

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Journal titleJournal of the American Chemical Society
Pages56775686; # of pages: 10
Subjectalpha tocopherol; cholesterol esterase; article; enzyme activity; hydrolysis; reaction analysis
AbstractThe noncompetitive and competitive hydrolyses of (2R,4'R,8'R)-α-tocopheryl acetate (RRR-α-TOAc, the acetate of natural vitamin E) and (2S,4'R,8'R)-α-tocopheryl acetate (SRR-α-TOAc) catalyzed by crude and pure bovine cholesterol esterase (BCE) and crude and pure porcine cholesterol esterase (PCE) have been studied at 37 °C. These two CE's are catalytically active toward tocopheryl acetates only in the presence of bile salts. The three 3α,7α,12α-trihydroxy bile salts, cholate, glycocholate and taurocholate, not only are effective activators of BCE and PCE, but also modulate the diastereoselectivities of these two enzymes in their hydrolyses of RRR- and SRR-α-TOAc in a characteristic manner. Rates of hydrolyses were much faster in the presence of a small quantity of dl- or l-dimyristoylphosphatidylcholine (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 salt/co-lipid ratio, nor by the purity of the enzyme. In noncompetitive experiments, the ratios of the BCE-catalyzed initial rates of hydrolyses of the diastereomeric acetates, V(i)(RRR)/V(i)(SRR), are 0.21, 1.5, and 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 concentrations of RRR- and SRR-α-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 taurocholate, 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, more probably, its phenolic product. Glycochenodeoxycholate, a 3α,7α-dihydroxy bile salt, activates PCE but not BCE. With the former enzyme, the diastereoselectivities, V(i)(RRR)/V(i)(SRR) are rather similar 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 salts modulate the diastereoselectivities of BCE and PCE by a direct effect on the protein which may involve 'refolding' the enzyme with a consequent change in the shape of the active site. It should be noted that the relevant chiral carbon in α-TOAc is separated by six bonds from the bond which is broken and that this chiral center must exert its influence prior to the rate-limiting step in the overall hydrolysis. If the bile salt modulating effect on CE-catalyzed reactions is to be exploited in organic syntheses, it is the competitive experiments which are the more important, and for these, the diastereoselectivity, V(i)(RRR)/V(i)(SSR), 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 dramatic 40-fold change in an enzyme's chiral selectivity is without precedent.
Publication date
AffiliationNational Research Council Canada (NRC-CNRC)
Peer reviewedYes
NPARC number21276709
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Record identifier27dea4a4-eccb-453d-9807-a55bd21a25eb
Record created2015-10-13
Record modified2016-05-09
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