Effects of site-directed mutagenesis at residues cysteine-31 and cysteine-184 on lecithin-cholesterol acyltransferase activity.
Native lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein, and LCAT in which either or both of the enzyme free cysteines had been replaced with glycine residues by site-directed mutagenesis, has been expressed in cultured Chinese hamster ovary cells stably transfected with the human LCAT gene. The mass of LCAT secreted, determined by immunoassay, did not differ in the native and mutant species. LCAT specific activity was also unchanged in the mutant species. In particular, the cysteine-free double mutant, in which Cys-31 and Cys-184 had both been replaced, was fully active in the synthesis of cholesteryl esters. This result is not consistent with a catalytic role for LCAT free cysteine residues. The classical inhibitor of LCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which strongly (89%) inhibited the native enzyme, had partial (45%) inhibitory activity with mutant enzyme species containing a single -SH residue, while the double mutant was not significantly inhibited by DTNB. These data are interpreted to suggest that Cys-31 and Cys-184 are vicinal both to each other and to the "interfacial binding site" at residues 177-182, and that DTNB exerts its effect by steric inhibition.
Francone
O L
OL
Cardiovascular Research Institute, University of California Medical Center, San Francisco 94143.
Fielding
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003545:Cysteine; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003545:Cysteine