NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

Abstract

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 C^α 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.

Scheme 1. Pyridoxal-5′-phosphate

Scheme 2. α-Deprotonation and Quinonoid Resonance Forms

Scheme 3. Tryptophan Synthase β-Site Reaction

Scheme 4. Formation of the 2AP Quinonoid Intermediate

Figure 1

(a) X-ray crystal structure of the tryptophan synthase α₂β₂ heterodimer, highlighting the β-subunit active site in red. (b) Cluster model of the β-subunit active site for first-principles geometry optimization and chemical shift calculations. Protein side chains are shown in wireframe, and the cofactor and substrates are in ball-and-stick. (c) Potential sites of protonation on and near the cofactor/substrate complex include (A) the βLys87 side chain, (B) the PLP phosphate group, (C) the PLP pyridine ring nitrogen, (D) the PLP phenolic oxygen, (E) the Schiff-base nitrogen, and (F,G) the substrate carboxylate. Shaded nuclei indicate sites for which experimental NMR chemical shifts are reported.

Scheme 5. Carbanionic Phenolic and Protonated Schiff Base Tautomers with NBO Partial Charges Indicated at C^α and C4′

Scheme 6. Quinonoid Phenolic and Protonated Schiff Base Tautomers with NBO Partial Charges Indicated at C^α and C4′

Figure 2

¹⁵N SSNMR CPMAS spectra of the microcrystalline TS 2AP quinonoid intermediate prepared with the following isotopic labeling: (a) natural abundance isotopomer concentration; (b) selectively ¹³C,¹⁵N enriched on the PLP cofactor; (c) ¹⁵N-enriched on the substrate l-Ser; (d) ¹⁵N-labeled on the substrate 2AP; and (e,f) selectively ¹⁵N-enriched at lysine ε-nitrogen side chain sites. Spectra (e) and (f) form an ¹⁵N{³¹P}-REDOR pair; both have a 25 ms echo period on ¹⁵N before detection, but (f) includes the application of dipolar dephasing to ³¹P. Their difference spectrum (Δ) allows for the selective observation of N^ε for the active site lysine side chain. Spectra acquired at 9.4 T (a–d), 14.1 T (e,f), and 8 kHz MAS; additional experimental details are given in the main text.

Figure 3

Variable-temperature ¹⁵N and ¹³C CPMAS spectra of the microcrystalline TS 2AP quinonoid intermediate prepared with 2,2′,3-¹³C₃,¹⁵N-PLP, ¹⁵N-Ser, and U-¹⁵N-TS. (a) Substantial temperature dependence is observed for the Schiff base nitrogen (blue dot). The large spectral feature at 330 ppm is a spinning sideband of the labeled amide backbone. (b) Temperature dependence is also observed for the PLP C3 site (red dot), which shows a resolved scalar coupling to C2. Spectra acquired at 9.4 T and 8 kHz MAS; additional experimental details are given in the main text.

Figure 4

¹³C SSNMR CPMAS spectra of the microcrystalline TS 2AP quinonoid intermediate prepared under (a) natural abundance isotopomer concentration; (b) selectively ¹³C,¹⁵N-enriched on the PLP cofactor; and (c) U-¹³C₃,¹⁵N-enriched on the substrate l-Ser. Spectra acquired at 9.4 T and 8 kHz MAS; additional experimental details are given in the main text.

Figure 5

³¹P SSNMR CPMAS spectrum of the microcrystalline TS 2AP quinonoid intermediate acquired at 9.4 T and 2 kHz MAS. The PLP phosphate group isotropic peak at 4.9 ppm is indicated by the arrow and that from F9 with the asterisk. A fit (red) to the sideband manifold in BrukerTopspin 3.0 allows for the extraction of the CSA principal axis components {δ₁₁, δ₂₂, δ₃₃} = {56.8, −7.1, −35.0} ppm for the PLP phosphate group and {δ₁₁, δ₂₂, δ₃₃} = {66.3, −11.0, −44.6} ppm for F9. The order of the spinning sidebands is given above each peak.

Figure 6

(a) Reduced-χ² comparing the experimental and first-principles isotropic chemical shifts for 30 geometry-optimized active-site models with varying protonation states (structures and labeling given in Scheme S1). Models with reduced-χ² greater than 1.75 can be excluded with over 95% confidence. (b) Reduced-χ² comparing the experimental and first-principles isotropic and anisotropic chemical shifts for the 10 best fast-exchange equilibrium models. For each optimized isotropic model (red), the corresponding population weighted CSA tensors are also compared to the experimental principal axis components for the substrate C′ (gray) and Schiff base N (blue) sites.

Figure 7

Protonation states and hydrogen-bonding interactions revealed by NMR crystallography in the tryptophan synthase β-subunit active site. The 2AP quinonoid intermediate is found to be a carbanionic species undergoing fast proton exchange between its (a) phenolic (81% occupancy) and (b) protonated Schiff base (19% occupancy) tautomeric forms. Several of the key hydrogen-bonding interactions to the cofactor and substrate are indicated, including the (standard) hydrogen bond between the PLP ring nitrogen and βSer377. Images rendered in UCSF Chimera.