Abstract
Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.