Adenosine 2‘-Monophosphate, 5‘-O-[S-(4-Succinimidylbenzophenone)- thiophosphate]:  A New Photoaffinity Label for the Coenzyme Site of Porcine NADP-Specific Isocitrate Dehydrogenase

Abstract

A new photoaffinity label, adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (2'-P-AMPS-Succ-BP), has been synthesized by an initial thiophosphorylation of 2'-AMP with PSCl(3) to form 2'-AMP-5'-thiophosphate (2'-AMP-5'-SP), followed by a coupling reaction of 2'-AMP-5'-SP with benzophenone-4-maleimide to produce 2'-P-AMPS-Succ-BP. This product and its precursor were characterized by thin-layer chromatography, (31)P NMR, phosphorus analysis, and electron-spray mass spectroscopy. 2'-P-AMPS-Succ-BP functions as a photoaffinity label of porcine NADP-specific isocitrate dehydrogenase. To obtain reaction with other amino acids, Cys269 and Cys379, the most reactive cysteines of this enzyme, were mutated to yield a double mutant enzyme (C269A/C379S) exhibiting comparable activity and kinetic parameters to those of wild-type enzyme. 2'-P-AMPS-Succ-BP inactivates C269A/C379S enzyme upon UV irradiation. The reaction exhibits a nonlinear relationship of k(inact) versus [2'-P-AMPS-Succ-BP] with K(R) = 12 microM and k(max) = 0.0275 min(-1). NADP, NADPH, or 2'-monophospho-adenosine 5'-diphosphoribose protects the enzyme against 2'-P-AMPS-Succ-BP inactivation. The ligand protection studies suggest that 2'-P-AMPS-Succ-BP binds to the porcine enzyme at the site best occupied by NADP/NADPH. The dimeric C269A/C379S isocitrate dehydrogenase incorporates 1.0 mol of 2'-P-[(35)S]AMPS-Succ-BP/mol enzyme dimer concomitant with complete loss of enzyme activity. The new photoaffinity label may be generally useful to identify important amino acid residues of NADP-specific enzymes.

Conjugation Reactions Involving Maleimides and Phosphorothioate Oligonucleotides

“…Thiophosphates, either mono-or diesters, are known to react with bromo-or iodoacetyl groups, 10,11 and this was reported to be the preferred reaction when PS-ONs and bifunctional linkers containing maleimide and haloacetyl groups were mixed. 12 Phosphorothioate monoesters and the ω-thiophosphate of nucleoside di-and triphosphates react with maleimides, 13−17 giving the corresponding Michael-type adduct. The Michael addition has been described as being more difficult in the case of thiophosphate diesters, especially in comparison with the thiol−maleimide reaction, but taking place to a non-negligible extent.…”

“…Phosphorothioate monoesters and the ω-thiophosphate of nucleoside di- and triphosphates react with maleimides, − giving the corresponding Michael-type adduct. The Michael addition has been described as being more difficult in the case of thiophosphate diesters, especially in comparison with the thiol–maleimide reaction, but taking place to a non-negligible extent.…”