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.
CITATION STYLE
Mitchell, D. A., Pendleton, L. C., & Deschenes, R. J. (2019). In vitro assays to monitor the enzymatic activities of zDHHC protein acyltransferases. In Methods in Molecular Biology (Vol. 2009, pp. 169–177). Humana Press Inc. https://doi.org/10.1007/978-1-4939-9532-5_13
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