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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA base in the peptidyl transferase center
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Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA base in the peptidyl transferase center

机译：肽基转移酶中心中pKa干扰的RNA碱基对稳定过渡态氧阴离子的证据

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摘要

The crystal structure of the ribosomal 50S subunit from Haloarcula marismortui in complex with the transition state analog CCdA-phosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an "oxyanion hole" to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000) Science 289, 920-929]. In the model, close proximity (3 A) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.
机译：滨海假单胞菌核糖体50S亚基的晶体结构与过渡态类似物CCdA-磷酸盐-嘌呤霉素（CCdApPmn）的复合物导致了一种机制性提议，其中核糖体活性位点中普遍被颠倒的A2451充当了“氧阴离子孔”以促进肽基转移酶反应[Nissen，P.，Hansen，J.，Ban，N.，Moore，PB，and Steitz，TA （2000）Science 289，920-929]。在该模型中，A2451 N3与CCdApPmn的非桥连氨基磷酸氧之间的紧密接近（3 A）表明，在四面体过渡态期间形成的羰基氧阴离子通过氢键合到质子化的A2451 N3上而得以稳定，其pKa必须被扰动实质上。我们通过使用化学保护和肽基转移抑制试验来表征A2451的N3与非桥联氨基磷酸氧之间假定的氢键的贡献。如果此推定的氢键起重要的热力学作用，则与50d核糖体亚基的CCdApPmn结合亲和力应强烈依赖于pH值，并且随着pH值降低，亲和力会增加。我们报告CCdApPmn在5.0和8.5之间的所有pH值上以实质上相等的亲和力结合50S核糖体。这些数据反对肽基转移的机制，在该机制中，具有接近中性pKa的残基稳定了过渡态含氧阴离子，至少达到了CCdApPmn准确模拟过渡态的程度。

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