Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism.

摘要: Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement phospholipase A2 transacylase activities enzyme using proteoliposome substrates following selective chemical modification serine, histidine, cysteine residues pure homogeneous LCAT indicated catalytic mechanism: HS-Cys-E-Ser-OH + in equilibrium HS-Cys-E-Ser-O-FA lysolecithin, FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH cholesterol-OH cholesterol-O-FA, where FA denotes acid. Modification 2 with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment ferricyanide inactivated but not activity. 1 serine residue phenylmethanesulfonyl fluoride histidine diethyl pyrocarbonate inhibited formation Proteoliposome protected both against inactivation. Lecithin alone activity inactivation Incubation native arachidonyl-CoA lecithin-apo-A-I resulted acylation three sites, only one which was stable neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- thioesters were demonstrable cases. No transfer arachidonic from iodoacetamide-modified occurred, indicating that fatty-acylated cannot directly esterify cholesterol. Cholesterol arachidonate formed upon incubation fluoride-modified arachidonyl-CoA.