Allosteric regulation of -reaction stage I in tryptophan synthase upon the -ligand binding

Tryptophan synthase (TRPS) is a bifunctional enzyme consisting of alpha- and beta-subunits that catalyzes the last two steps of L-tryptophan (L-Trp) biosynthesis. The first stage of the reaction at the beta-subunit is called beta-reaction stage I, which converts the beta-ligand from an internal aldimine [E(Ain)] to an alpha-aminoacrylate [E(A-A)] intermediate. The activity is known to increase 3-10-fold upon the binding of 3-indole-D-glycerol-3 '-phosphate (IGP) at the alpha-subunit. The effect of alpha-ligand binding on beta-reaction stage I at the distal beta-active site is not well understood despite the abundant structural information available for TRPS. Here, we investigate the beta-reaction stage I by carrying out minimum-energy pathway searches based on a hybrid quantum mechanics/molecular mechanics (QM/MM) model. The free-energy differences along the pathway are also examined using QM/MM umbrella sampling simulations with QM calculations at the B3LYP-D3/aug-cc-pVDZ level of theory. Our simulations suggest that the sidechain orientation of beta D305 near the beta-ligand likely plays an essential role in the allosteric regulation: a hydrogen bond is formed between beta D305 and the beta-ligand in the absence of the alpha-ligand, prohibiting a smooth rotation of the hydroxyl group in the quinonoid intermediate, whereas the dihedral angle rotates smoothly after the hydrogen bond is switched from beta D305-beta-ligand to beta D305-beta R141. This switch could occur upon the IGP-binding at the alpha-subunit, as evidenced by the existing TRPS crystal structures.