Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase.
Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase.
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腺苷 2-单磷酸，5-O-[S-(4-琥珀酰亚胺基二苯甲酮)-硫代磷酸]：猪 NADP 特异性异柠檬酸脱氢酶辅酶位点的新光亲和标记。
DOI：
10.1021/bc049722w
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Colman,RobertaF
中科院分区：
文献类型：
--
作者：
Lee,Peychii;Colman,RobertaF
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 PSCl3to 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,31P 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 ofkinactversus [2‘-P-AMPS-Succ-BP] withKR= 12 μM andkmax= 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-[35S]AMPS-Succ-BP/mol enzyme dimer concomitant withcompleteloss of enzyme activity. The new photoaffinity label may be generally useful to identify important amino acid residues of NADP-specific enzymes.
