Mitchell David A, Pendleton Laura C, Deschenes Robert J
Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
Methods Mol Biol. 2019;2009:169-177. doi: 10.1007/978-1-4939-9532-5_13.
A family of zDHHC protein acyltransferase (PAT) enzymes catalyze the S-palmitoylation of target proteins via a two-step mechanism. The first step involves transfer of palmitate from the palmitoyl-CoA donor to the active site cysteine of the zDHHC PAT enzyme, releasing reduced CoA (CoASH). In the second step, the palmitoyl-PAT intermediate thioester reacts with a cysteine side chain within the target substrate to produce the palmitoylated substrate product or, in the absence of a protein substrate, the palmitoyl-PAT intermediate thioester is hydrolyzed and releases palmitate. Formation and resolution of the palmitoyl-PAT intermediate complex (autopalmitoylation) is measured using a coupled enzyme system that monitors the production of CoASH via reduction of NAD by the α-ketoglutarate dehydrogenase complex. This assay can be used to isolate and characterize modulators of autopalmitoylation and is scalable to high-throughput screening (HTS). A second fluorescence-based assay is described that monitors the hydrolysis of the palmitoyl-PAT thioester linked intermediate by thin-layer chromatography using a palmitoyl-CoA analog, BODIPY-C12:0-CoA, as a substrate. These two assays provide a methodology to quantify the first enzymatic step of the two-step zDHHC PAT reaction.
zDHHC蛋白酰基转移酶（PAT）家族的酶通过两步机制催化靶蛋白的S-棕榈酰化。第一步涉及将棕榈酸从棕榈酰辅酶A供体转移到zDHHC PAT酶的活性位点半胱氨酸上，释放出还原型辅酶A（CoASH）。第二步，棕榈酰-PAT中间硫酯与靶底物内的半胱氨酸侧链反应生成棕榈酰化底物产物，或者在没有蛋白质底物的情况下，棕榈酰-PAT中间硫酯被水解并释放出棕榈酸。使用耦合酶系统测量棕榈酰-PAT中间复合物的形成和分解（自身棕榈酰化），该系统通过α-酮戊二酸脱氢酶复合物还原NAD来监测CoASH的产生。该测定法可用于分离和表征自身棕榈酰化的调节剂，并且可扩展用于高通量筛选（HTS）。还描述了另一种基于荧光的测定法，该方法使用棕榈酰辅酶A类似物BODIPY-C12:0-CoA作为底物，通过薄层色谱法监测棕榈酰-PAT硫酯连接的中间体的水解。这两种测定法提供了一种方法来量化两步zDHHC PAT反应的第一步酶促反应。
