Program in Chemical Biology, ‡Department of Chemistry, and §Department of Pharmacology, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
ACS Chem Biol. 2013 Sep 20;8(9):1912-7. doi: 10.1021/cb400380s. Epub 2013 Jul 25.
2-Bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a nonselective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.
2-溴十六烷酸，或 2-溴棕榈酸，在近 50 年前被引入作为脂质代谢的非选择性抑制剂。最近，2-溴棕榈酸重新成为蛋白质 S-棕榈酰化的通用抑制剂。在这里，我们通过合成和应用点击反应活性的类似物来研究 2-溴棕榈酸的细胞靶标。在细胞中，2-溴棕榈酸被转化为 2-溴棕榈酰-CoA，但效率低于游离棕榈酸。一旦与 CoA 结合，探针的反应活性就会大大增强。重要的是，2-溴棕榈酸和 2-溴棕榈酰-CoA 都标记 DHHC 棕榈酰基转移酶（PATs），即催化蛋白质 S-棕榈酰化的酶。富集的 2-溴棕榈酸靶目标的质谱分析鉴定出 PAT 酶、转运蛋白和许多棕榈酰化蛋白，没有观察到对 CoA 依赖性酶的偏好。这些数据质疑 2-溴棕榈酸（或 2-溴棕榈酰-CoA）是否通过抑制蛋白酰基转移酶或通过直接共价竞争抑制棕榈酸掺入来阻断 S-棕榈酰化。总的来说，这些发现强调了 2BP 的混杂反应性，并验证了可点击的 2BP 类似物作为多种膜相关酶的基于活性的探针的有效性。
