Single-molecule FRET reveals how urea paradoxically increases the activity of an enzyme.

Proteins often harness large-scale motions of domains and subunits to promote their function. The enzyme adenylate kinase (AK) has served as a playground for testing the potential relation between conformational dynamics and enzymatic activity. Efficient catalysis is ensured by large-scale domain motion that leads to the enclosure of the bound substrates ATP and AMP. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we identify two key mechanisms for this phenomenon: first, urea promotes the open conformation of the enzyme, which aids in the proper positioning of the substrates. Second, urea also decreases AMP affinity, thus facilitating a more efficient progression towards the catalytic ternary complex and leading to an unexpected outcome where reducing substrate affinity benefits turnover. In instances of little or no significant substrate inhibition by AMP, the enzyme shows no activation by urea. Our results demonstrate the important interplay between chemical steps such as binding and conformational dynamics in the activity of enzymes, which can be identified using state-of-the-art tools, such as single-molecule fluorescence spectroscopy.