IDENTIFICATION OF ADENINE BINDING DOMAIN PEPTIDES OF THE NADP(-SITE WITHIN PORCINE HEART NADP(+)-DEPENDENT ISOCITRATE DEHYDROGENASE() ACTIVE)

Photoaffinity labeling with [2'-P-32]2N(3)NADP(+) and [P-32]2N(3)NAD() was used to identify two overlapping tryptic and chymotryptic genera ted peptides within the adenine binding domain of NADP(+)-dependent is ocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of ID H prevented insertion, Photoincorporation of 2N(3)NAD(+) inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [P-32]2N(3) NAD(+) and [2'-P-32]2N(3)NAD(P)+ showed saturation effects with appare nt K(d)s of 20 and 14 mu M (+/-12%), respectively. The efficiency of p hotoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [P-32]8N(3)ATP and [P -32]2N(3)ATP but With saturation effects observed at lower affinity, W ith all radiolabeled probes reduction of photoinsertion was effected b est by the addition of NADP(+) followed by NAD(+) and then ATP, indica ting that photoinsertion with all the probes was within the NADP(+) bi nding site. Isolation of [P-32]2N(3)NAD(+) and [2'-P-32]2N(3)NADP(+) p hotolabeled peptides by use of immobilized boronate and immobilized Al 3+ chromatography, respectively, followed by HPLC purification resulte d in the identification of overlapping peptides corresponding to Ile(2 44)-Arg(249) and Leu(121)-Arg(133) (tryptic fragments) and Lys(243)-Hi s(248) and Leu(121)-His(135) (chymotryptic fragments). Trp(125) and Tr p(245) were identified as the sites of photoinsertion based on these r esidues not being detectable on sequencing, the lack of chymotryptic c leavage at these residues, and the decreased rate of trypsin digestion at nearby Lys(243) and Lys(127). Sequence analysis of [(32)]8N(3)ATP and [P-32]2N(3)ATP photolabeled peptides gave essentially the same pep tide regions being photolabeled but at much lower efficiency, indicati ng that the effects of ATP on IDH activity are dependent on competitio n for the same site.