ROLE OF GLUTAMIC-ACID RESIDUE-154, RESIDUE-155, AND RESIDUE-165 OF LECITHIN-CHOLESTEROL ACYLTRANSFERASE IN CHOLESTEROL ESTERIFICATION AND PHOSPHOLIPASE A(2) ACTIVITIES

Previous studies have shown that cholesterol esterification activity b y lecithin:cholesterol acyltransferase (LCAT) is progressively inhibit ed as up to three acidic acid residues are chemically modified. The pu rpose of this study was to determine whether three glutamic acid resid ues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid bind ing region of apoE, were necessary for enzymatic activity. Site-direct ed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholi pase A(2) (PLA(2)) assays. Cholesterol esterification for all mutant c onstructs (11-26 nmol CE/h/mu g) was similar to or greater than that o f wild type LCAT (16 nmol CE/h/mu g), except for a triple mutant, in w hich glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/mu g). PLA(2) activity followed a similar trend. There wa s a significant decrease in the cholesterol esterification to PLA(2) a ctivity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residue s 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol bind ing at the active site of the enzyme.