Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth at the atomic scale. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction at runtime has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis "in action" by "mix-and-inject serial crystallography". Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis beta-lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.