Human plasma lecithin-cholesterol acyltransferase. The vicinal nature of cysteine 31 and cysteine 184 in the catalytic site

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins up which the enzyme acts to form the majority of cholesteryl ester in human plasma. We proposed a covalent catalytic mechanism of action for LCAT (Jauhiainen, M., and Dolphin, P.J. (1986) J. Biol. Chem. 261, 7032-7034) in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols we have probed the geometry of the catalytic site. p-Aminophenylarsendichloride noncompetitively inactivates cholesterol esterification (K(i) = 0.23 mM) by LCAT via alkylation of both catalytic cysteine residues. This reagent does not significantly inactivate lecithin cleavage b LCAT. Full enzyme activity is restored by treatment with 2,3-dimercapto-1-propanesulfonic acid. Treatment of LCAT with p-bromo-acetylaminophenylarsenoxide blocks the subsequent incorporation of diisopropyl fluorophosphate and iodoacetamide and inactivates both cholesterol esterification and lecithin cleavage. These activities are not restored following 2,3-dimercapto-1-propanesulfonic acid treatment. However, the reduced cysteine thiols are regenerated and can catalyze cholesteryl arachidonate formation from arachidonyl-CoA. The control reagent, bromoacetylaniline, which lacks the sulfhydryl-reactive arsenical moiety, does not inactivate LCAT nor is this reagent incorporated into the LCAT protein. We conclude that the two catalytic cysteine residues of LCAT (Cys³¹ and Cys¹⁸⁴) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 Å. The additional residue alkylated by the bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.