Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions.
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 (31)P 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 (31)P 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 (His(26)) or lysine (Lys(33)) side-chain.
Ruman
Tomasz
T
Rzeszów University of Technology, Faculty of Chemistry, Department of Biochemistry and Biotechnology, 6 Powstańców Warszawy Ave., 35-959 Rzeszów, Poland. tomruman@prz.edu.pl
Długopolska
Karolina
K
Jurkiewicz
Agata
A
Rut
Dagmara
D
Fraczyk
Tomasz
T
Cieśla
Joanna
J
Leś
Andrzej
A
Szewczuk
Zbigniew
Z
Rode
Wojciech
W
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007477:Ions / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000737:chemistry*; D010756:Phosphoric Acids / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000577:Amides; D000596:Amino Acids; D007477:Ions; D010710:Phosphates; D010756:Phosphoric Acids; D011994:Recombinant Proteins; D006639:Histidine; C011067:phosphoramidic acid; D013940:Thymidylate Synthase; D014357:Trypsin; D008239:Lysine; C035638:thiophosphoric acid