Exploring Reactive Conformations of Coenzyme A during Binding and Unbinding to Pyruvate Formate–Lyase

Pyruvate formate–lyase (PFL) is a glycyl radical enzyme that converts pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. Recently, we showed that the acetylation of the PFL active site in the first half-reaction induces subtle conformational changes, leading to the opening of a potential channel for CoA entry. Entry of CoA into the active site is crucial for the second half-reaction, involving the acetyl transfer to CoA, and the completion of the catalytic cycle. Using steered molecular dynamics (SMD) simulations, performed on acetylated and nonacetylated monomeric PFL model systems, we first of all investigate the possible entry/exit pathways of CoA with respect to the active site through the previously identified channel. We then perform umbrella sampling simulations on multiple snapshots from SMD trajectories as well as unrestrained molecular dynamics simulations starting from the final structures obtained from entry SMD, with a view to identifying possible bound states of CoA in the near vicinity of the active site. Detailed study of the unrestrained dissociation processes reveals the presence of stable and reactive bound states of CoA close to the active site, one of which is in an ideal position for triggering the second half-reaction. Examination of the spatial distributions associated with the reactive bound states allows us to discuss the free energy barriers. Umbrella sampling, performed on snapshots from unrestrained dynamics confirms the above findings. The significance of the results for the catalysis are discussed for both acetylated and nonacetylated systems.