Enzymes are inherently dynamic molecules that sample multiple conformations on a wide range of timescales, many of which are essential for biological function. However, structural characterisations of enzymes typically focus on the most populated conformation, even though single-point mutations often lead to large changes in activity without detectable structural differences. Here, we investigate how intrinsic conformational dynamics dictate enzymatic activity and inhibitor potency using the class I histone deacetylase HDAC8 as a model system.
Using solution NMR spectroscopy, in particular, methyl-TROSY–based CPMG relaxation dispersion experiments, we quantitatify millisecond conformational exchange dynamics in wild-type HDAC8 and several single-point mutants. We show that reduced enzymatic activity, decreased inhibitor affinity, and shortened inhibitor residence times can all be explained by changes in the rate constants and populations of intrinsically sampled conformations. Importantly, these dynamic parameters are obtained independently by NMR and thus independently of enzymatic or binding assays, demonstrating that functional changes are encoded directly in the intrinsic dynamics of the enzyme.
Our analysis identifies distinct conformational states that are selectively involved in substrate binding, catalysis, and product release, including states that are sparsely populated and not represented in available crystal structures. By combining NMR-derived exchange kinetics with biochemical assays and kinetic modelling, we show that differences in intrinsic dynamics alone are sufficient to quantitatively account for the altered activities and inhibitor potencies of HDAC8 mutants.
Together, these results demonstrate that precise structures alone are often insufficient to explain the functional impact of missense mutations. Instead, intrinsic enzyme dynamics provide a framework for understanding enzymatic regulation, reduced activity in missense mutants, and drug potency, with broader implications for enzyme characterisation and inhibitor design.