Abstract
Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.
Highlights
Enzymes are molecular machines that bind substrates provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates
The values we find for closing (0.18 nM À 1 s À 1) and opening (1 s À 1), as well as the associated equilibrium constant (3 nM) almost exactly match published values for AP5A binding[23] and affinity[21] measured in bulk assays for Escherichia coli adenylate kinase (AdK)
The possibility of studying native-state conformational energies using mechanical single-molecule methods allows us to quantitatively determine the energetic drive of conformational changes of an enzyme induced by substrate binding
Summary
Enzymes are molecular machines that bind substrates provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. We have employed a high-resolution optical tweezers setup to study the mechanical coupling of substrate binding and lid closing in the enzyme adenylate kinase (AdK). We used single-molecule optical tweezers to directly measure the substrate-dependent forces that drive AdK into a closed conformation. Our data directly show that closing and opening of both lids is cooperative, as the molecules always transition between the fully open and fc states This interpretation is supported by a mutant where we attach our handles to only one lid (ATP lid) and the CORE domain (for details see Methods). We find conformational changes of only half the size (ca. 0.9 nm) albeit with identical kinetics (Supplementary Fig. 4)
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