Mechanism of ribosome catalyzed peptide bond formation

这项研究计划的目标是确定核糖体如何催化进化上保守的 和生物学上必不可少的肽键形成反应。这是一个长期的生物化学目标， 然而，若干重要问题仍未得到解答。核糖体在化学上对催化作用有贡献吗？ 它是否利用金属离子作为催化辅因子？它使用的是一般的酸催化剂还是一般的碱催化剂？如果 两者，质子转移是协调的还是逐步的？如果两者都不是，那么采用了什么其他策略？它是如何 利用P-位点tRNA末端的顺式二醇促进反应？做出了哪些贡献 底物辅助催化研究建议中概述了一系列平行、互补、但 从根本上不同的方法，将揭示这种生物学上的基本反应的机制。 这些实验利用了各种技术，包括合成有机化学，酶动力学， 生物化学和结构生物学。区域特异性，过渡态手性， 生长的肽基链和活性位点内溶剂原子的身份将使用 活性位点抑制剂的一系列更复杂的产品。底物辅助催化的本质 并通过测量P位反应动力学来检验金属离子在衬底活化中的作用 含有位点特异性化学取代的tRNA。化学过渡态的性质和 参与反应的每个原子上的键级和电荷的相对程度将由下式确定： 动力学同位素效应分析利用一系列非天然氨基酸，具有广泛的pKa 值，将确定肽基转移酶反应的布朗斯台德系数。这将提供 支持或反对一般碱或一般酸催化机理的进一步证据。酶的功能是 与过渡态的结合比基态更紧密。确定电荷分布 过渡态将使得有可能开发针对核糖体的紧密结合抗生素， 改进已经用于治疗细菌感染的抗生素。 2-3句子摘要核糖体负责制造所有生物中的所有蛋白质。这是一个 治疗细菌感染的主要药物靶点。在这个研究项目中获得的信息 将带来对抗疾病的改良抗生素。

The goal of this research program is to determine how the ribosome catalyzes the evolutionarily conserved and biologically essential reaction of peptide bond formation. This has been a long term biochemical goal, yet several significant questions remain unanswered. Does the ribosome contribute chemically to catalysis? Does it utilize metal ions as catalytic cofactors? Does it utilize a general acid or a general base catalyst? If both, is the proton transfer concerted or step-wise? If neither, what other strategy is employed? How does it utilize the cis-diol at the P-site tRNA terminus to promote the reaction? What contribution is made by substrate-assisted catalysis? Outlined in the research proposal is a series of parallel, complementary, yet fundamentally different approaches that will reveal the mechanism of this biologically essential reaction. These experiments utilize a full gamut of techniques including synthetic organic chemistry, enzyme kinetics, biochemistry and structural biology. The regiospecificity, transition state chirality, the exit pathway for the growing peptidyl chain, and the identity of solvent atoms within the active site will be tested using a progressively more sophisticated series of active site inhibitors. The nature of substrate assisted catalysis and the role of metal ions in substrate activation will be tested by measuring the reaction kinetics of P-site tRNAs containing site specific chemical substitutions. The nature of the chemical transition state and the relative degree of bond order and charge on each atom involved in the reaction, will be determined by kinetic isotope effect analysis. Utilizing a series of unnatural amino acids with a broad spectrum of pKa values, the Bronsted coefficients of the peptidyl transferase reaction will be determined. This will provide further evidence for or against general base or general acid catalytic mechanisms. Enzymes function by binding more tightly to their transition states than their ground states. Determining the charge distribution of the transition state will make it possible to develop tight binding antibiotics against the ribosome and improve upon the antibiotics already utilized to treat bacterial infection. 2-3 sentence summary. The ribosome is responsible for making all the proteins in all living things. It is a primary drug target for the treatment of bacterial infection. The information gained in this research program will lead to improved antibiotics for combating disease.