Interplay between conformational dynamics and substrate binding regulates enzymatic activity: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. We utilize this phenomenon to decipher the involvement of conformational dynamics in the mechanism of action of the enzyme. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Further, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results allow us to define a complete kinetic scheme that includes the open/close transitions of the enzyme and to unravel the important interplay between conformational dynamics and chemical steps, a general property of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.