We have “watched” a key glycolytic enzyme evolve as the bacteria that expressed it adapted to a new environment.  Our study builds on a long-term evolution experiment, where 12 replicate bacterial populations were evolved for >60,000 generations.  Adaptive evolution resulted in eight separate beneficial mutations in the gene pykF. Each mutation increased the fitness of the population in which it occurred.  PykF encodes the enzyme pyruvate kinase, a key allosterically regulated enzyme within the energy production pathway, glycolysis.  Biochemical studies demonstrate that the mutations altered the function of each evolved pyruvate kinase relative to the ancestor, and each other.  Despite dramatically different enzyme functions, the structure of each mutant was very similar to that of the wild-type enzyme, as judged by x-ray crystallography, small angle x-ray scattering and analytical ultracentrifugation. This was a conundrum.  Thus, we turned to microfluidic assisted hydrogen/deuterium exchange coupled with mass spectrometry to 1) determine whether protein dynamics are important for function in the WT enzyme, and 2) gauge how point mutations in the protein sequence affected the dynamics of the evolved enzymes.