John J, Sohmen R, Feuerstein J, Linke R, Wittinghofer A, Goody R S
Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, Heidelberg, FRG.
Biochemistry. 1990 Jun 26;29(25):6058-65. doi: 10.1021/bi00477a025.
A method is described for the convenient preparation of substantial quantities of nucleotide-free p21 or of 1:1 complexes with nucleotides other than GDP. The nucleotide-free protein has been used for kinetic studies of the binding of GDP and GTP, making use of the fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP and -GTP. Stopped-flow studies have led to the formulation of a two-step binding mechanism for both GDP and GTP, involving initial rapid but weak binding of the nucleotide followed by a relatively slow (10-20 s-1 at 25 degrees C; 3-5 s-1 at 5 degrees C) quasi-irreversible isomerization reaction. By use of a nonequilibrium competition method, guanosine and GMP have been shown to interact weakly but significantly with p21 (dissociation constants of 153 and 29 microM, respectively). The presence of guanosine or GMP at the active site of p21 leads to a marked stabilization of p21 against spontaneous denaturation when compared with the nucleotide- and nucleoside-free protein.
本文描述了一种便捷的方法，可大量制备无核苷酸的p21或与除GDP之外的核苷酸形成的1:1复合物。无核苷酸的蛋白质已用于GDP和GTP结合的动力学研究，利用了荧光类似物3'-(甲基氨茴酰基)-2'-脱氧-GDP和-GTP。停流研究得出了GDP和GTP的两步结合机制，包括核苷酸最初的快速但弱结合，随后是相对缓慢的（25℃时为10-20 s-1；5℃时为3-5 s-1）准不可逆异构化反应。通过使用非平衡竞争方法，已证明鸟苷和GMP与p21有弱但显著的相互作用（解离常数分别为153和29 microM）。与无核苷酸和无核苷的蛋白质相比，p21活性位点存在鸟苷或GMP会导致p21对自发变性有明显的稳定性。
