Department of Chemistry and Biochemistry, University of California , Santa Barbara, California 93106-9510, United States.
Biochemistry. 2013 Mar 12;52(10):1669-76. doi: 10.1021/bi301284f. Epub 2013 Feb 27.
One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.
酶促进高能中间体的一个最显著的例子发生在 DNA 修饰和修复酶中，其中单个碱基围绕脱氧核糖-磷酸主链旋转 180°并进入活性位点。虽然终态已经得到了广泛的描述，但实验技术尚未提供碱基翻转过程的完整描述以及酶的作用。C5 胞嘧啶甲基转移酶 M.HhaI 协调一系列相互作用的 DNA 和酶重排，以有效地将靶标胞嘧啶从 DNA 螺旋中翻转出来。我们试图了解单个氨基酸在碱基翻转过程中的作用。我们的结果表明，M.HhaI 在催化环关闭之前启动碱基翻转，并利用催化环中的保守丝氨酸 85 加速翻转并保持 DNA 骨架的扭曲。在碱基翻转后形成 DNA 螺旋内特定接触的丝氨酸 87 不参与翻转过程或维持催化有效复合物。在催化环的底部，甘氨酸 98 充当铰链，允许环的构象动态变化，突变到丙氨酸会抑制闭环的稳定。我们的结果说明了酶如何协同利用众多的、远距离的残基将底物识别转化为催化。
