Wydział Chemii

Tomasz Ruman

K. Długopolska

A. Jurkiewicz

D. Rut

Tomasz Frączyk

J. Cieśla

A. Leś

Zbigniew Szewczuk

Wojciech Rode

2010

Bioorganic Chemistry

38

74-80

10.1016/j.bioorg.2009.11.002

Naukowa

Angielski

Artykuł

In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of ³¹P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of ³¹P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His²⁶) or lysine (Lys³³) side-chain.

NMR, Thiophosphate, Tiophosphoramidate, Thiophosphorylation, Thymidylate synthase

https://doi.org/10.1016/j.bioorg.2009.11.002

http://www.elsevier.com