Importance of the free sulfhydryl groups of lecithin-cholesterol acyltransferase for its sensitivity to oxidative inactivation

Lecithin-cholesterol acyltransferase (LCAT) of human plasma is known to be highly susceptible to oxidative inactivation, although the mechanism of thi s inactivation is unknown. We tested the hypothesis that the high sensitivi ty of the enzyme is due to the derivatization of its two free SH groups fla nking the active site pocket. Modification of the SE-I groups with a revers ible inhibitor protected the enzyme against oxidative inactivation. Mutagen esis of either of the cysteines to glycine increased the resistance of the enzyme, which retained 46% of activity in presence of 150 muM Cu2+ compared to only 27% of the activity retained by the wild type enzyme (WT). Replace ment of both the cysteines with glycines resulted in retention of over 65% activity. Cysteine replacement similarly protected the enzyme from inactiva tion by the oxidized substrate. Chicken LCAT, which has only one cysteine ( Cys(26)), was more resistant than the human enzyme. Introduction of an addi tional cysteine corresponding to the second cysteine in human LCAT (N184C) resulted in increased susceptibility of chicken enzyme (87% loss of activit y in presence of 150 muM Cu2+, compared to 55% loss in WT). Substitution of the lone cysteine with glycine (C26G) resulted in a more resistant enzyme, which lost <40% activity under the same conditions. These results show tha t the primary targets of the oxidizing agents or the products of oxidation are the SH groups of the enzyme, whose derivatization leads to steric inhib ition of the activity. (C) 2000 Elsevier Science B.V. All rights reserved.