Abstract
Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compounds for the detection of the enzymatic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these molecules gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compounds for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaption to high-throughput screening of small molecule inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.
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