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 this inactivation is unknown. We tested the hypothesis that the high sensitivity of the enzyme is due to the derivatization of its two free SH groups flanking the active site pocket. Modification of the SH groups with a reversible inhibitor protected the enzyme against oxidative inactivation. Mutagenesis of either of the cysteines to glycine increased the resistance of the enzyme, which retained 46% of activity in presence of 150 microM Cu(2+), compared to only 27% of the activity retained by the wild type enzyme (WT). Replacement of both the cysteines with glycines resulted in retention of over 65% activity. Cysteine replacement similarly protected the enzyme from inactivation by the oxidized substrate. Chicken LCAT, which has only one cysteine (Cys(26)), was more resistant than the human enzyme. Introduction of an additional cysteine corresponding to the second cysteine in human LCAT (N184C) resulted in increased susceptibility of chicken enzyme (87% loss of activity in presence of 150 microM Cu(2+), 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 that the primary targets of the oxidizing agents or the products of oxidation are the SH groups of the enzyme, whose derivatization leads to steric inhibition of the activity.
Wang
K
K
Department of Medicine, Rush Medical College, 1653 West Congress Parkway, Chicago, IL 60612, USA.
Subbaiah
P V
PV
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002645:Chickens; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014162:Transfection
D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003545:Cysteine; D005998:Glycine