In the histone deacetylase 8 (HDAC8) enzyme, which is a potential target of cancer, wild type and mutant HDAC8s show very similar structures when bound to substrate/inhibitors. Therefore, static structures have not been able to adequately explain observed changes in biochemical activity and substrate/inhibitor binding affinities for various HDAC8 mutants. Using methyl-TROSY based CPMG relaxation dispersion experiments, we show that the dynamic sampling of different conformations of HDAC8 dictate the enzymatic activity, inhibitor affinity, and inhibitor residence time. Our analysis of HDAC8 also dissects the functional role of the conformations sampled, where specific conformations are responsible for substrate/inhibitor binding, catalysis, and product dissociation.