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标题：Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A2 activities
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作者：Jingchuan Wang ; Jeanine A. DeLozier ; Abraham K. Gebre 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：1998
卷号：39
期号：1
页码：51-58
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed 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 phospholipase A₂ (PLA₂) assays. Cholesterol esterification for all mutant constructs (11–26 nmol CE/h/μg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/μg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/μg). PLA₂ activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA₂ activity 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 residues 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 binding at the active site of the enzyme.— Wang, J., J. A. DeLozier, A. K. Gebre, P. J. Dolphin, and J. S. Parks. Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A₂ activities. J. Lipid Res. 1998. 39: 51–58.
关键词：COS cells ; apolipoprotein E ; apolipoprotein A-I ; recombinant HDL mutagenesis ; glutamic acid ; LCAT ; enzyme activity