Inactive but Essential: The Role of the Inactive State of E49 in the Mechanism of the Alpha Subunit of Tryptophan Synthase and Its Stand-Alone Blueprint <i>Zm</i> BX1

The stand-alone version of the alpha subunit of tryptophan synthase (TrpA), <i>Zm</i>BX1, catalyzes the retro-aldol cleavage of indole-3-glycerol phosphate (IGP) at a catalytic efficiency that is approximately 144,000 times higher than that of isolated <i>Zm</i>TrpA. Available X-ray crystal structures of <i>Zm</i>BX1 and several TrpAs revealed identical overall structures as well as active site geometries, showing high flexibility of the catalytic E49 in both cases. Based on the crystallographic data, E49 was found to adopt an active state in which the carboxylate group is close to IGP for promoting the retro-aldol cleavage as well as an additional inactive state whose catalytic function was unclear. In this work, by using a combination of Molecular Dynamics (MD) simulations and cluster model DFT calculations, we rationalize the effect of the active/inactive conformation of the catalytic E49, as well as how L2 containing the other catalytically relevant residue D60 affects catalysis. The higher levels of retro-aldol activity observed for <i>Zm</i>BX1 are attributed to its dual ability to adopt not only active states of the catalytic E49 crucial for retro-aldol cleavage but also inactive states that position E49 in a noncatalytic orientation for disfavoring the reverse aldol reaction back to IGP after product formation. Our combined MD and QM studies elucidate the mechanistic interplay between conformational changes and catalytic steps in <i>Zm</i>BX1 and TrpA enzymes. This study highlights the importance of optimizing the conformational changes and chemical steps along the catalytic itinerary for altering and/or improving enzymatic function.