Different metastable substrate conformations would be expected to experience different local environments for the aspartic acid, which in turn could impact the pKa values. We also note that a similar conformational selection mechanism has been described for the Kemp eliminases HG3 and KE07. It must be noted, however, that, unlike HG3 and KE07, the active site of our Kemp eliminase was originally generated 28 using a single-mutation minimalist design that may mimic the actual emergence of new enzymes during natural evolution. Our results therefore suggest the relevance of the conformational selection in the evolutionary optimization of primordial enzymes.