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

• Published in: Angewandte Chemie International Edition 2016-01, Vol.55 (4), p.1350-1354
• Main Authors: Young, Robert P., Caulkins, Bethany G., Borchardt, Dan, Bulloch, Daryl N., Larive, Cynthia K., Dunn, Michael F., Mueller, Leonard J.
• Format: Article
• Language: English
• Subjects: enzymes; homogeneous catalysis; ligases; NMR spectroscopy; proteins
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201508898

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 17O 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. Enzymatic reactions: Several intermediates in the catalytic cycle of tryptophan synthase were characterized by 17O quadrupole central‐transition NMR spectroscopy. The picture shows the 17O solution‐state NMR spectrum of the kinetically competent α‐aminoacrylate intermediate superimposed on the β‐subunit active site.