کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1942452 1052614 2012 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک دانش گیاه شناسی
پیش نمایش صفحه اول مقاله
Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups
چکیده انگلیسی

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [3H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.


► The hydrophobic binding pocket of the carnitine/acylcarnitine carrier has been studied.
► The protein residues that bind the acyl groups have been identified by mutagenesis.
► Val-25, Pro-78, Val-82, Met-85, Cys-89 bind acyl-moieties of acylcarnitines.
► Ala-276, Ala-279, Cys-283 are involved in other steps of translocation.
► These residues face the protein central cavity forming an hydrophobic half shell.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Bioenergetics - Volume 1817, Issue 5, May 2012, Pages 697–704
نویسندگان
, , , , ,