The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site substrate analogues, N-(4’-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in 19F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformation states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

The acid–base chemistry that drives catalysis in pyridoxal-5′-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP’s role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first 15N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional 13C, 15N, and 31P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4′-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the β-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the β-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed β-site. We conclude that hydrophobic molecules can freely diffuse between the α- and β-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αβ-catalytic cycle. Finally, while most TS structures show βPhe280 partially blocking the tunnel between the α- and β-sites, new structures show an open tunnel, suggesting the flexibility of the βPhe280 side chain. Flexible docking studies and MD simulations confirm the dynamic behavior of βPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5′-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and β-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3′-phosphate and l-Ser to l-Trp. In this process, the roles played by β-site residues proximal to the PLP cofactor have not yet been fully established. βGln114 is one such residue. To explore the roles played by βQ114, we conducted a detailed investigation of the βQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet–visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the β-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and βQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of βGln114, βAsn145, and βArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (βT state) and closed (βR state) conformations of the β-subunit and consequently alter the distribution of intermediates along the β-subunit catalytic path.