Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3 beta-thiol as a fatty acyl acceptor.

Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3 beta-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 beta-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin/thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The Km for thiocholesterol was 203.6 microM with a Vmax of 5.3 nmol thiocholesteryl ester formed/h per microgram. The Km for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 microM with a Vmax of 8.8 nmol cholesteryl ester formed/h per microgram. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3 beta-SH group of thiocholesterol forming a cholesteryl thioester. Although the 3 beta-SH group is not as good a terminal acceptor as the 3 beta-OH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester.