Huang Kevin S, Carrasco Nicolas, Pfund Emmanuel, Strobel Scott A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Biochemistry. 2008 Aug 26;47(34):8822-7. doi: 10.1021/bi800299u. Epub 2008 Aug 2.
The ribosomal peptidyl transferase is a biologically essential catalyst responsible for protein synthesis. The reaction is expected to proceed through a transition state approaching tetrahedral geometry with a specific chirality. To establish that stereospecificity, we synthesized two diastereomers of a transition state inhibitor with mimics for each of the four ligands around the reactive chiral center. Preferential binding of the inhibitor that mimics a transition state with S chirality establishes the spatial position of the nascent peptide and the oxyanion and places the amine near the critical A76 2'-OH group on the P-site tRNA. Another inhibitor series with 2'-NH 2 and 2'-SH substitutions at the critical 2'-OH group was used to test the neutrality of the 2'-OH group as predicted if the hydroxyl functions as a proton shuttle in the transition state. The lack of significant pH-dependent binding by these inhibitors argues that the 2'-OH group remains neutral in the transition state. Both of these observations are consistent with a proton shuttle mechanism for the peptidyl transferase reaction.
核糖体肽基转移酶是一种对蛋白质合成至关重要的生物催化剂。预计该反应通过接近四面体几何结构且具有特定手性的过渡态进行。为了确定这种立体特异性，我们合成了一种过渡态抑制剂的两种非对映异构体，其中围绕反应性手性中心的四个配体均有模拟物。优先结合模拟具有S手性过渡态的抑制剂，确定了新生肽和氧负离子的空间位置，并使胺靠近P位点tRNA上关键的A76 2'-OH基团。另一系列在关键的2'-OH基团处有2'-NH₂和2'-SH取代的抑制剂，用于测试2'-OH基团的中性，前提是如果羟基在过渡态中充当质子穿梭体，则如预测的那样。这些抑制剂缺乏明显的pH依赖性结合，这表明2'-OH基团在过渡态中保持中性。这两个观察结果均与肽基转移酶反应的质子穿梭机制一致。
