Bharadwaj M, Bizzozero O A
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131-5221, USA.
J Neurochem. 1995 Oct;65(4):1805-15. doi: 10.1046/j.1471-4159.1995.65041805.x.
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 nonenzymatic 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 degrees 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 approximately 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.
P0是周围神经系统髓磷脂的主要蛋白质，在细胞质中的半胱氨酸153处发生棕榈酰化。为深入了解P0酰化的机制，研究了P0和含半胱氨酸153的合成八肽的体外棕榈酰化。用[3H]棕榈酰辅酶A孵育周围神经系统髓磷脂膜或分离出的P0，会使该蛋白质发生特异性标记，这表明该反应是非酶促的。在孵育前将分离出的P0煮沸15分钟或向反应混合物中加入坐骨神经匀浆，均不影响标记脂肪酸掺入P0，这证实了该反应的非酶促性质。化学脱酰化后，P0的棕榈酰化速率更高，表明原来的位点被重新酰化。此外，胰蛋白酶消化和肽图谱分析表明，体外酰化的位点与神经切片中的相同，这表明该反应具有生理意义。用[14C]棕榈酰辅酶A孵育时，包含天然P0酰化位点（I150RYCWLRR157）的合成肽在半胱氨酸残基处也会自发酰化。因此，非酶促转酰化反应不需要蛋白质的完整性。在pH 7.4和37℃条件下，肽的棕榈酰化遵循二级反应（k2 = 246±6 M-1 min-1），可能是肽硫醇盐攻击棕榈酰辅酶A中高反应性硫酯键的双分子亲核取代反应。根据阿伦尼乌斯图计算的活化能约为2千卡/摩尔，远低于酶催化转酰化反应的活化能。最后，另外两个P0肽（V121PTRYG126和K109TSQVTL115）以及各种不相关的含硫醇化合物，包括半胱氨酸、谷胱甘肽、加压素酸（CYFQNC）和甲壳类动物心脏活性肽（PFCNAFTGC），均未发生自动酰化。这些结果表明，IRYCWLRR肽代表一种特殊的结构基序和/或具有一些化学特征，使得反应能够自发发生。
