Nemat-Gorgani M, Wilson J E
Arch Biochem Biophys. 1986 Nov 15;251(1):97-103. doi: 10.1016/0003-9861(86)90055-x.
8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).
8-叠氮基-ATP可作为大鼠脑己糖激酶（ATP：D-己糖6-磷酸转移酶，EC 2.7.1.1）的底物。在这种可光活化的ATP类似物存在下对己糖激酶进行辐照会导致该酶失活。先前已证明能竞争性抑制核苷酸结合的ATP和6-磷酸己糖（葡萄糖-6-磷酸、1,5-脱水葡萄糖醇-6-磷酸）可保护该酶免受过光失活的影响；其他6-磷酸己糖则不能。先前已证明能增强核苷酸结合的己糖（葡萄糖、甘露糖）也能防止光失活；其他己糖则不能。己糖和6-磷酸己糖的这些作用可以根据先前所示由这些配体结合导致并影响核苷酸结合位点特征的构象变化来解释（M. Baijal和J. E. Wilson（1982年）《生物化学与生物物理学报》218卷，513 - 524页）。用胰蛋白酶对该酶进行有限切割产生三个主要片段，分子量约为10K、40K和50K，据认为它们代表该酶内的主要结构域（P. G. Polakis和J. E. Wilson（1984年）《生物化学与生物物理学报》234卷，341 - 352页）。在14C标记的叠氮基-ATP存在下对已光失活的该酶进行胰蛋白酶切割，发现10K和40K结构域有明显的标记，已知它们分别源自N端和C端区域。40K结构域的标记受配体影响的方式与这些配体防止光失活的有效性相对应，而10K结构域的标记不受这些相同配体的影响。得出的结论是，40K结构域包含核苷酸底物的结合位点。更精细的二维肽图谱技术表明，主要的标记位点是一个分子量约为20K的肽段，位于40K结构域的中部和/或C端区域。10K结构域的标记归因于叠氮基-ATP与已知位于脑己糖激酶N端的疏水序列的非特异性相互作用（P. G. Polakis和J. E. Wilson（1985年）《生物化学与生物物理学报》236卷，328 - 337页）。
