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Enzyme activity varies with temperature. Unlike small-molecule catalysts, the structural ensembles of enzymes can change substantially with temperature, but it is unclear how this modulates temperature dependent activity. Here, multi-temperature X-ray crystallography was used to record structural changes from -20C to 40C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes on the reaction coordinate. Inhibitors, substrates and active site loops increasingly populated catalytically competent conformations as temperature increased. These changes occurred even in temperature ranges where kinetic measurements showed roughly linear Arrhenius/Eyring behavior, where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy/enthalpy values vary with temperature. Our results indicate a critical role for temperature dependent atomic-resolution structural data in interpreting temperature dependent kinetic data from enzymatic systems. 

Serine proteases have been proposed to dynamically sample inactive and active conformations, but direct evidence at atomic resolution has remained elusive. Using nuclear magnetic resonance (NMR), we identified a single residue, D164, in exfoliative toxin A (ETA) that acts as a molecular “switch” to regulate global dynamic sampling. Mutations at this site shift the balance between inactive and active states, correlating directly with catalytic activity. Beyond identifying this dynamic switch, we demonstrate how it works in concert with other allosterically coupled sites to rationally control enzyme movements and catalytic function. This study provides a framework for linking conformational dynamics to function and paves the way for engineering enzymes, in particular, proteases, with tailored activities for applications in medicine and biotechnology.