Myelin P₀ Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

Abstract

P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153‐containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl‐CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl‐CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second‐order reaction (k2 = 246 ± 6 M−1 min−1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl‐CoA. The activation energy calculated from the Arrhenius plot is ∼2 kcal/mol and much lower than that of enzyme‐catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol‐containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.

The Canonical DHHC Motif Is Not Absolutely Required for the Activity of the Yeast S-acyltransferases Swf1 and Pfa4

“…It has been shown that, under certain conditions, the uncatalyzed palmitoylation reaction requires only five times the activation energy than the PAT-catalyzed reaction (40). This could mean that just approaching the two substrates could produce reaction rates that allow the detection of palmitoylated substrates in vivo.…”

COVALENT BINDING OF 2-PHENYLPROPIONYL-S-ACYL-COA THIOESTER TO TISSUE PROTEINS IN VITRO

“…Sallustio et al (2000) demonstrated that covalent binding of nafenopin to human liver proteins is directly associated with formation of a nafenopin acyl-CoA thioester intermediate. A number of studies on protein fatty acylation have shown that endogenous acyl-CoAs, including palmitoyl-CoA and arachidonoyl-CoA, can react nonenzymatically with sulfhydryl groups on proteins and peptides in vitro in a time-and concentration-dependent fashion (Bharadwaj and Bizzozero, 1995;Duncan and Gilman, 1996). Our recent studies with 2-phenylpropionic acid (2-PPA 3 ) demonstrated that 2-phenylpropionyl-S-acyl-CoA (2-PPA-CoA) was able to acylate glutathione sulfhydryl to form 2-PPA-S-acyl-glutathione at a rate that was approximately 70-fold more rapid than the similar reactions with 2-PPA-1-O-acyl glucuronides .…”

Studies on the Reactivity of Clofibryl-S-Acyl-CoA Thioester with Glutathione in Vitro

“…It was shown that the free amine of glycine, even under conditions in which the amine is protonated (pH 7.5), was acylated by salicyl-CoA to form salicyluric acid. A number of studies have been conducted to characterize the nonenzymatic acylation of protein sulfhydryls by endogenous acyl-CoA derivatives in vitro (Bharadwaj and Bizzozero, 1995;Yamashita et al, 1995;Duncan and Gilman, 1996). In such experiments, endogenous acyl-CoA derivatives, including palmitoyl-CoA and arachidonoyl-CoA, have been shown to react spontaneously with cysteine-containing proteins and peptides to form thioester conjugates in a time-and concentration-dependent fashion.…”