Solution‐State ¹⁷O 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 aprobe in NMR spectroscopy. Here,w ed emonstrate the first use of solution-state 17 Oquadrupole central-transition NMR spectroscopyt oc haracterizee nzymatic intermediates under conditions of active catalysis.I nt he 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase,reactions of the a-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 substratecofactor p-bonding network, particularly at the adjacent acarbon site.

Functional stability of water wire–carbonyl interactions in an ion channel

“…The potential of 17 O NMR for elucidating functional understanding of biological chemistry has long been recognized (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). Preliminary results from 17 O-labeled L10, L12, and L14 in the presence of double occupancy K + have been reported using low field spectroscopy showing that 17 O spectroscopy would be a much more sensitive tool for such studies than 15 N spectroscopy (34).…”

NMR spectroscopy up to 35.2 T using a series-connected hybrid magnet

“…The relaxation of spin > 1/2 nuclei from the quadrupolar interaction is multi-exponential and the behavior of the slow relaxing component is analogous to that of the central-transition of quadrupolar nuclei in solids. The quadrupole central-transition (QCT) line width in the slow tumbling regime has a strong field dependence, as demonstrated recently by the variable-field studies of 17 O-enriched oxalate bound to chicken ovotransferrin (total molecular weight ~ 80 kDa) [32, 33] and pyruvate kinase (240 kDa) [34] up to 21.1 T. Fig. 10 shows 17 O QCT spectra of [3,5,6- 17 O]-D-glucose in glycerol solution at different magnetic fields.…”

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…One approach that exploits quadrupolar relaxation and has been successful for the study of quadrupolar nuclei in the context of biological macromolecules (including 17 O, 27 Al, and 51 V) is quadrupole central transition (QCT) NMR (48, 158–160), which utilizes relaxation properties of the central transition at high magnetic fields (i.e. the slow tumbling regime) to obtain narrow lines.…”

“…the slow tumbling regime) to obtain narrow lines. The combination of high magnetic fields and narrow lines allows for the observation of fine structural details and applications to larger biological systems (158), as demonstrated in 17 O QCT experiments to study the catalytic mechanism of the enzyme Tryptophan synthase (48). …”

“…The group at NIMS in Japan has demonstrated measurements at 24 T on the spin 3/2 nuclei 35 Cl and 37 Cl (44, 45), as well as 17 O (1, 23). One of the fields with greatest potential for growth with the advent of magnetic fields over 1GHz is 17 O NMR (39, 46), a critical element in biology which could serve as an exquisite probe for a variety of mechanistic and structural questions, including enzyme catalysis, pharmaceuticals analysis, protein structure and hydration (47, 48), as well as materials/MOF applications mentioned above. To date 17 O has not been extensively exploited due to its extremely low natural abundance and the often prohibitive cost of isotopic enrichment.…”