Bao Letian, Forster Anthony C
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
J Biol Chem. 2025 Jul;301(7):110336. doi: 10.1016/j.jbc.2025.110336. Epub 2025 Jun 3.
Peptide bond formation and peptidyl release are catalyzed at the peptidyl transferase center of the 50S subunit of the 70S ribosome. Proposed catalytic mechanisms at the peptidyl transferase center are based on structures of model substrates bound to the 50S and the 70S. Yet, peptidyl transfer and release reactions catalyzed by the 50S are slower by >3 orders of magnitude than those of the 70S. Here, we obtained a near-physiological rate of peptide bond formation with puromycin catalyzed by the 50S in 33% methanol at 37 °C, and fast rates were even attained in aqueous solution using 20% PEG. Interestingly, methanol, not PEG, accelerated the reaction by stimulating substrate binding just to the 50S P site. In addition, we obtained fast peptidyl release model reactions catalyzed by tRNA or Cytosine-Cytosine-Adenine (CCA) trinucleotide on the 50S in 30% acetone. However, PEG did not enable the release reaction, suggesting different mechanisms for release and peptide bond formation. The now-comparable peptidyl transfer rates of the 50S and 70S under aqueous conditions strengthen mechanistic proposals, give credence to hypothetical progenitor ribosomes before evolution of the 30S and will aid mechanistic investigations with model substrates or ancestral subsets of the ribosome.
肽键形成和肽基释放是在70S核糖体50S亚基的肽基转移酶中心催化的。肽基转移酶中心的催化机制是基于与50S和70S结合的模型底物的结构提出的。然而，50S催化的肽基转移和释放反应比70S的反应慢3个数量级以上。在这里，我们在37℃下于33%甲醇中获得了由50S催化的与嘌呤霉素形成肽键的近生理速率，并且在使用20%聚乙二醇（PEG）的水溶液中甚至达到了快速反应速率。有趣的是，是甲醇而非PEG通过仅刺激底物与50S P位点的结合来加速反应。此外，我们在30%丙酮中获得了由tRNA或胞嘧啶-胞嘧啶-腺嘌呤（CCA）三核苷酸在50S上催化的快速肽基释放模型反应。然而，PEG不能促成释放反应，这表明释放和肽键形成的机制不同。现在在水性条件下50S和70S相当的肽基转移速率加强了机制推测，证实了在30S亚基进化之前假设的原始核糖体，并将有助于使用模型底物或核糖体的祖先亚基进行机制研究。
