Solution-State (17)O Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase

Abstract

Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.

Keywords: NMR spectroscopy; enzymes; homogeneous catalysis; ligases; proteins.

Figure 1

¹⁷O QCT NMR spectra of the E(Q₃)_indoline intermediate in TS at multiple magnetic fields. The enzyme-bound intermediates are marked with asterisks. Total number of transients and experiment times: at 11.7 T, 2.4 × 10⁶ transients and 22 hours; at 14.1 T, 2.2 × 10⁶ transients and 20 hours; at 16.4 T, 6.4 × 10⁶ transients and 59 hours. The spectra are displayed with 100 Hz line broadening applied. Experiments were conducted at 3±2°C in pH 7.8 buffered aqueous solution. Additional experimental details are provided in the SI.

Figure 2

¹⁷O quadrupole central transition NMR spectra of the E(AA)(BZI) intermediate in TS at multiple fields. The enzyme-bound intermediates are marked with asterisks. Experiments measured at 14.1 and 16.4 T were acquired using a triple echo-sequence to improve observation of the enzyme-bound peaks by attenuating free substrate/product signal. Total number of transients and experiment times: at 11.7 T, 7.9 × 10⁶ transients and 72 hours; at 14.1 T, 3.3 × 10⁶ transients and 40 hours; at 16.4 T, 12.9 × 10⁶ transients and 156 hours. The spectra are displayed with 100 Hz line broadening applied. Experiments were conducted at 3±2°C in pH 7.8 buffered aqueous solution. Additional experimental details are provided in the SI.

Figure 3

Model for first principles calculations of chemical shifts in the β subunit enzyme active site of tryptophan synthase. Side chains are shown in wireframe and the PLP-substrate as ball and stick.

Scheme 1

An abridged catalytic cycle for the TS β-subunit reaction.

Scheme 2

Exchange between tautomeric forms of the indoline quinonoid intermediate.