Balbo Paul B, Bohm Andrew
Department of Biochemistry, Tufts School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Structure. 2007 Sep;15(9):1117-31. doi: 10.1016/j.str.2007.07.010.
We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.
我们报告了通过将催化所需的天冬氨酸154突变为丙氨酸而捕获的与ATP和五个残基的聚腺苷酸（poly(A)）形成复合物的酵母聚腺苷酸聚合酶（PAP）的1.8埃结构。该酶经历了显著的结构域移动，并呈现出一种封闭构象，底物与所有三个聚合酶结构域之间存在广泛相互作用。底物和31个埋藏水分子都被封闭在一个两端开放的中央腔内。对四个PAP突变体进行了详细的动力学分析，并展示了利用CTP对这些突变体进行腺苷酰转移（正向）、焦磷酸解（反向）和核苷酸转移反应的研究。结果支持了一个模型，即聚腺苷酸和正确的核苷酸MgATP的结合都会诱导构象变化，导致形成稳定的、封闭的酶状态。讨论了数据的热力学考虑因素，因为它们与结构域封闭、底物特异性以及PAP所采用的催化策略有关。
