Bioconjugate chemistry

Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase.

PMID 15898734


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.

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Adenosine 2′-monophosphate, from yeast