DFT-Assisted Evolution of In-Cell Protein-Acetylation Catalyst

Post-translational modifications (PTMs) of proteins play a crucial role in dynamically regulating cellular biological processes. Chemical catalysts have emerged as versatile tools for probing and modulating PTMs. A hydroxamic acid-thiol conjugated (HXA-SH) catalyst, mBnA, functionally mimics acetyltransferases by uniquely activating endogenous acetyl-coenzyme A (Ac-CoA) to promote regioselective lysine acetylation of targeted proteins in living cells. However, its in-cell acetylation activity has remained insufficient for furnishing biological function (2.5% yield for epigenetically valuable histone protein), and the key factors governing catalytic activity have yet to be fully elucidated. Here, we identify the rate-determining step of the catalytic reaction using Density Functional Theory (DFT) calculations, revealing a pivotal proton-shuttling process that neutralizes the charge-separated intermediate involving the catalyst, substrate amine, and a water molecule. Guided by these mechanistic insights, we develop an improved HXA-SH catalyst, mHXA-pOMe, which facilitates the proton-shuttling process. The mHXA-pOMe catalyst exhibits a significant increase in in-cell histone acetylation yield, reaching 20% without the need for exogenous acetyl donors. These findings align with enzymatic mechanisms and provide a strategic foundation for advancing synthetic protein acetylation catalysts capable of modulating cellular functions.