A view on phosphate ester photochemistry by time-resolved solid state NMR. Intramolecular redox reaction of caged ATP

Abstract

The light-driven intramolecular redox reaction of adenosine-5'-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the alpha-, beta-, and gamma-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is 2.4 +/- 0.2 h(-1) at 245 K. The proton released in the reaction binds to the triphosphate moiety of the nascent ATP, causing the upfield shifts of the 31P resonances. Analyses of the reaction kinetics indicate that bond cleavage and proton release are two sequential processes in the solid state, suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the reaction. The beta-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are interpreted in terms of chemical exchange processes, hydrogen bonding of the beta-phosphate oxygen atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP. Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis of ATP-dependent enzymes by NMR spectroscopy in rotating solids.

Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling

“…Current approaches to investigate enzyme mechanism at the atomic level [X-ray crystallography (4,51–53), 31 P-NMR spectroscopy (54,55), ab initio QM/MM simulations (56–60), etc.] provide highly significant key observations, but with each having their inherent limitations requiring integration of multiple methods.…”

High‐Resolution Studies of Uniformly 13C,15N‐Labeled RNA by Solid‐State NMR Spectroscopy

“…[8] In addition, solid-state NMR spectroscopy emerges as a powerful tool for the time-resolved study of macromolecular folding and catalysis. [9][10][11] By varying the temperature of the frozen sample, structural and chemical transitions can be selectively trapped or monitored in real time. [11][12][13] Herein we apply solid-state NMR spectroscopy for atomic studies on RNA using a cUUCGg tetraloop hairpin as a model.…”

“…[9][10][11] By varying the temperature of the frozen sample, structural and chemical transitions can be selectively trapped or monitored in real time. [11][12][13] Herein we apply solid-state NMR spectroscopy for atomic studies on RNA using a cUUCGg tetraloop hairpin as a model. We have recently characterized this hairpin in solution and obtained a refined high-resolution structure (RMSD = 0.3 ).…”

Hochauflösende Festkörper‐NMR‐Spektroskopie an vollständig 13C,15N‐markierter RNA

“…[8] Zusätzlich konnten erste Experimente zeigen, dass sich die Festkörper-NMR-Spektroskopie zu einer leistungsfähigen Methode für zeitaufgelöste Studien der makromolekularen Faltung und Katalyse entwickelt. [9][10][11] Dabei können strukturelle und chemische Übergänge des Proteins durch ¾nderung der Temperatur selektiv fixiert ("eingefroren") oder zeitaufgelöst verfolgt werden. [11][12][13] Hier untersuchen wir Ribonukleinsäuren (RNA) durch Festkörper-NMR-Spektroskopie in atomarer Auflösung, wobei wir eine cUUCGg-Schlaufen-RNA als Modell verwenden.…”

“…[9][10][11] Dabei können strukturelle und chemische Übergänge des Proteins durch ¾nderung der Temperatur selektiv fixiert ("eingefroren") oder zeitaufgelöst verfolgt werden. [11][12][13] Hier untersuchen wir Ribonukleinsäuren (RNA) durch Festkörper-NMR-Spektroskopie in atomarer Auflösung, wobei wir eine cUUCGg-Schlaufen-RNA als Modell verwenden. Vor kurzem haben wir diese Schlaufen-RNA in Lösung untersucht und eine hochaufgelöste Struktur (RMSD = 0.3 ) bestimmt.…”