Department of Chemistry and Biochemistry, University of California , Santa Barbara, California 93106, United States.
Biochemistry. 2013 Mar 12;52(10):1677-85. doi: 10.1021/bi3012912. Epub 2013 Feb 27.
A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.
定量理解构象转变如何促进酶催化和特异性仍然是一个基本挑战。我们使用了一系列生物物理方法来揭示模型 DNA 胞嘧啶甲基转移酶 M.HhaI 的酶-底物复合物的几种瞬态结构。多维、横向弛豫优化的核磁共振（NMR）实验表明，M.HhaI 与非同源和同源 DNA 序列具有相同的构象。高亲和力的类似物模式需要形成一组蛋白质-DNA 相互作用，这些相互作用驱动靶碱基从螺旋到活性位点的翻转。缺乏这些相互作用的非同源底物会经历缓慢的碱基翻转，并且催化环的荧光跟踪证实了 NMR 证据，即在碱基翻转和随后的催化环闭合之前存在松散的、非特异性结合模式。这种缓慢的翻转转变定义了非同源序列甲基化的限速步骤。此外，我们还提供了沿碱基翻转途径的中间物的光谱证据，该中间物已经被预测但从未被观察到。这些发现提供了关于构象重排如何用于平衡特异性和催化效率的重要细节。
