Grammer J C, Kuwayama H, Yount R G
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Biochemistry. 1993 Jun 8;32(22):5725-32. doi: 10.1021/bi00073a001.
The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (t1/2 approximately 5 days, 0 degree C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co(III)phenanthroline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of the heavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.
ATP在骨骼肌肌球蛋白上的嘌呤结合位点已用2-叠氮基腺苷二磷酸（2-N3ADP）进行了光亲和标记。通过两条重链反应性硫醇（半胱氨酸-697和半胱氨酸-707）与三价钴菲咯啉的络合作用，2-N3ADP在活性位点被稳定捕获（半衰期约5天，0℃）。光掺入仅发生在重链的23-kDa NH2-末端胰蛋白酶片段中。用胰蛋白酶和枯草杆菌蛋白酶对肌球蛋白的光标记亚片段1进行广泛的连续消化，得到一系列经HPLC纯化的标记肽段。对八个光标记肽段的序列和放射性标记分析均表明，色氨酸-130是唯一被标记的残基。该标记位点证实了早期用非核苷酸ADP类似物2-[(4-叠氮基-2-硝基苯基)-氨基]乙基二磷酸（NANDP）进行的光标记研究，该类似物也标记了色氨酸-130 [冈本洋, & 扬特, R. G. (1985) 《美国国家科学院院刊》82, 1575 - 1579]。2-N3ADP和NANDP结构的比较表明，如果这两种类似物以类似于ATP反式构象的伸展构象结合到活性位点，它们的叠氮基团可以重叠。
