Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates

Both enzymatic and autocatalytic mechanisms have been proposed to account f or protein thioacylation (commonly known as palmitoylation). Acyl-CoA bindi ng proteins (ACBP) strongly suppress non-enzymatic thioacylation of cystein yl-containing peptides by long-chain acyl-CoAs. At physiological concentrat ions of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acyla tion is expected to be too slow to contribute significantly to thioacylatio n of signaling proteins in mammalian cells (Leventis et al., Biochemistry 3 6 (1997) 5546-5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characteriz ed using G-protein alpha-subunits as a substrate (Dunphy et al., J. Biol. C hem., 271 (1996) 7154-7159), was capable of thioacylating short lipid-modif ied cysteinyl-containing peptides. The minimum requirements for substrate r ecognition were a free cysteine thiol adjacent to a hydrophobic lipid ancho r, either myristate or farnesyl isoprenoid. PAT activity displayed specific ity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhi bited enzymatic thioacylation of a myristoylated peptide or G-protein alpha -subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl-CoA in vitro and is likel y to represent the predominant mechanism of thioacylation in vivo. (C) 2000 Esevier Science B.V. All rights reserved.