Urea as a Protein LID Opener: Overcoming Substrate Inhibition in Adenylate Kinase by Tuning Conformational Dynamics

Abstract

Enzymes are designed to accelerate vital chemical reactions by multiple orders of magnitude. Many of them harness large-scale motions of domains and subunits to promote their activity. Studying structural dynamics is hence essential in order to decipher how protein machines function. Combined with H2MM, a photon-by-photon hidden Markov model analysis, single-molecule FRET studies on the enzyme adenylate kinase (AK) recently revealed very fast domain motions, two orders of magnitude faster than the turnover of the enzyme. AK may use numerous cycles of conformational rearrangement in order to find a relative orientation of its substrates that is optimal for the chemical reaction, the interconversion of adenine nucleotides. In the present work we studied the impact of urea on the domain motions of AK, particularly those of its ATP-binding LID-domain. Although urea has been commonly used as a protein denaturant, it is reported that in minor amounts it actually enhances the catalytic activity of AK. We show that this activation is caused by an effect on substrate inhibition. High concentrations of the substrate AMP decrease the rate of LID-domain opening, disturb the balance between open and closed conformations and reduce turnover. Minor amounts of urea partially revert this effect by opening the domains and allow the enzyme to regain activity. Importantly, in absence of inhibitory concentrations of AMP, the activity of AK is not enhanced by urea and can even be reduced, although urea here again favors the open conformation This suggests a delicate balance between domain closure dynamics and substrate binding, which might not be a unique feature of AK, and is likely employed by a multitude of enzymes to regulate their activity.