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Acyl‐coenzyme A :cholesterol acyltransferase 1 – significance of single‐nucleotide polymorphism at residue 526 and the role of P ro347 near the fifth transmembrane domain
Author(s) -
Huang LiHao,
Nishi Koji,
Li Song,
Ho Thomas,
Dong Ruhong,
Chang Catherine C. Y.,
Chang TaYuan
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12739
Subject(s) - sterol o acyltransferase , biochemistry , acyltransferases , active site , enzyme , transmembrane domain , biology , chemistry , cholesterol , amino acid , lipoprotein , biosynthesis
Acyl‐coenzyme A :cholesterol acyltransferases ( ACAT s), which are members of the membrane‐bound O ‐acyltransferase family, catalyze the conversion of cholesterol to cholesteryl esters. Mammals have two isoenzymes: ACAT 1 and ACAT 2. Both enzymes are drug targets for treating human diseases. ACAT 1 is present in various cell types. It contains nine transmembrane domains ( TMD s), with the active site His460 located within TMD 7, and the active site Asn421 located within the fourth large cytoplasmic loop. In human ACAT 1, a single‐nucleotide polymorphism exists for residue 526: the codon is either CAG for G ln, or CGG for A rg. G ln526/Arg526 is present within the C ‐terminal loop. Its biochemical significance is unknown. In addition, within the C ‐terminal half of ACAT 1, numerous residues conserved with those of ACAT 2 are present; the functions of these conserved residues are largely unknown. Here, we performed single‐substitution mutagenesis experiments to investigate the roles of individual residues present in the C ‐terminal loop, including G ln526/Arg526, and the eight conserved Pro residues located near/in various TMD s. The results show that the enzyme activity of ACAT 1 with G ln526 is less active than that of ACAT 1 with Arg526 by 40%. In addition, several residues in the C ‐terminal loop are important for maintaining proper ACAT 1 protein stability. Other results show that P ro347 plays an important role in modulating enzyme catalysis. Overall, our results imply that the CAG / CGG polymorphism can be utilized to perform ACAT 1 activity/human disease susceptibility studies, and that Pro347 located near TMD 5 plays an important role in modulating enzyme catalysis.