Single-molecule analysis of ligand-dependent regulation of HDAC8 catalysis

Histone deacetylase 8 (HDAC8) is a highly flexible enzyme whose activity is tightly linked to conformational dynamics and ligand interactions. Using single-molecule electronic nanocircuits, we directly monitored HDAC8's catalytic motions in real time to uncover how substrate modifications and small-molecule activators regulate its activity. Earlier work demonstrated that minor structural changes in inhibitors and substrates lead to pronounced differences in conformational subsets and kinetic pathways, highlighting HDAC8's sensitivity to ligand architecture. Building on this foundation, we now show how substrate modifications, including a trifluoroacetyl group and a Boc cap, accelerate catalysis by reshaping pre-chemical conformational steps, while the allosteric activator ACT synergistically stabilizes productive states. Statistical analysis of dwell-time distributions reveals that trifluoroacetyl substitution enhances the chemical step, whereas Boc and ACT simplify the conformational search, flattening the free-energy landscape and increasing turnover frequency nearly ten-fold. Together, these studies establish a unified framework in which multi-site ligand interactions dynamically tune HDAC8 catalysis. This approach offers mechanistic insight into enzyme regulation and suggests strategies for designing bifunctional ligands or activators that exploit cooperative conformational control.