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Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase.
Bioconjug Chem. 2005 May-Jun; 16(3):650-9.BC

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.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15898734

Citation

Lee, Peychii, and Roberta F. Colman. "Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a New Photoaffinity Label for the Coenzyme Site of Porcine NADP-specific Isocitrate Dehydrogenase." Bioconjugate Chemistry, vol. 16, no. 3, 2005, pp. 650-9.
Lee P, Colman RF. Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase. Bioconjug Chem. 2005;16(3):650-9.
Lee, P., & Colman, R. F. (2005). Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase. Bioconjugate Chemistry, 16(3), 650-9.
Lee P, Colman RF. Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a New Photoaffinity Label for the Coenzyme Site of Porcine NADP-specific Isocitrate Dehydrogenase. Bioconjug Chem. 2005 May-Jun;16(3):650-9. PubMed PMID: 15898734.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase. AU - Lee,Peychii, AU - Colman,Roberta F, PY - 2005/5/19/pubmed PY - 2005/9/9/medline PY - 2005/5/19/entrez SP - 650 EP - 9 JF - Bioconjugate chemistry JO - Bioconjug Chem VL - 16 IS - 3 N2 - 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. SN - 1043-1802 UR - https://www.unboundmedicine.com/medline/citation/15898734/Adenosine_2'_monophosphate_5'_O_[S__4_succinimidylbenzophenone__thiophosphate]:_a_new_photoaffinity_label_for_the_coenzyme_site_of_porcine_NADP_specific_isocitrate_dehydrogenase_ L2 - https://doi.org/10.1021/bc049722w DB - PRIME DP - Unbound Medicine ER -