Identification of Adenine Binding Domain Peptides of the NADP+ Active Site within Porcine Heart NADP+-Dependent Isocitrate Dehydrogenase
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- 作者：Banumathi Sankaran ; Ashok J. Chavan ; and Boyd E. Haley
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：October 22, 1996
- 年：1996
- 卷：35
- 期：42
- 页码：13501 - 13510
- 全文大小：482K
- 年卷期：v.35,no.42(October 22, 1996)
- ISSN：1520-4995
文摘
Photoaffinity labeling with[2'-32P]2N3NADP+ and[32P]2N3NAD+ was used toidentify twooverlapping tryptic and chymotryptic generated peptides within theadenine binding domain of NADP+-dependent isocitrate dehydrogenase (IDH). Photolysis was requiredfor insertion of radiolabel, and priorphotolysis of photoprobes before addition of IDH prevented insertion.Photoincorporation of2N3NAD+inhibited the enzymatic activity of IDH. Photolabeling of IDH withboth [32P]2N3NAD+ and[2'-32P]2N3NADP+ showed saturation effects with apparentKds of 20 and 14 
M (±12%), respectively.The efficiencyof photoincorporation at saturation of binding sites was determined tobe about 50%. Also, photolabelingwas observed with [32P]8N3ATP and[32P]2N3ATP but with saturation effectsobserved at lower affinity.With all radiolabeled probes reduction of photoinsertion waseffected best by the addition of NADP+followed by NAD+ and then ATP, indicating thatphotoinsertion with all the probes was within theNADP+binding site. Isolation of[32P]2N3NAD+ and[2'-32P]2N3NADP+photolabeled peptides by use ofimmobilized boronate and immobilized Al3+ chromatography,respectively, followed by HPLC purificationresulted in the identification of overlapping peptides corresponding toIle244-Arg249 andLeu121-Arg133(tryptic fragments) and Lys243-His248 andLeu121-His135 (chymotryptic fragments).Trp125 and Trp245 wereidentified as the sites of photoinsertion based on these residues notbeing detectable on sequencing, thelack of chymotryptic cleavage at these residues, and the decreased rateof trypsin digestion at nearbyLys243 and Lys127. Sequence analysis of[32P]8N3ATP and[32P]2N3ATP photolabeled peptidesgaveessentially the same peptide regions being photolabeled but at muchlower efficiency, indicating that theeffects of ATP on IDH activity are dependent on competition for thesame site.
M (±12%), respectively.The efficiencyof photoincorporation at saturation of binding sites was determined tobe about 50%. Also, photolabelingwas observed with [32P]8N3ATP and[32P]2N3ATP but with saturation effectsobserved at lower affinity.With all radiolabeled probes reduction of photoinsertion waseffected best by the addition of NADP+followed by NAD+ and then ATP, indicating thatphotoinsertion with all the probes was within theNADP+binding site. Isolation of[32P]2N3NAD+ and[2'-32P]2N3NADP+photolabeled peptides by use ofimmobilized boronate and immobilized Al3+ chromatography,respectively, followed by HPLC purificationresulted in the identification of overlapping peptides corresponding toIle244-Arg249 andLeu121-Arg133(tryptic fragments) and Lys243-His248 andLeu121-His135 (chymotryptic fragments).Trp125 and Trp245 wereidentified as the sites of photoinsertion based on these residues notbeing detectable on sequencing, thelack of chymotryptic cleavage at these residues, and the decreased rateof trypsin digestion at nearbyLys243 and Lys127. Sequence analysis of[32P]8N3ATP and[32P]2N3ATP photolabeled peptidesgaveessentially the same peptide regions being photolabeled but at muchlower efficiency, indicating that theeffects of ATP on IDH activity are dependent on competition for thesame site.