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 以及形成多肽键的生物本质反应。这是一个长期的生化目标， 然而，几个重要的问题仍然没有得到回答。核糖体对催化有化学作用吗？ 它是否利用金属离子作为催化辅因子？它使用的是普通酸催化剂还是普通碱催化剂？如果 两者，质子转移是协调的还是逐步的？如果两者都不是，那么还会采用什么策略？它是怎么做到的 利用P-位tRNA末端的顺式二醇来促进反应？做出什么贡献的是 底物辅助催化？研究提案中概述的是一系列平行的、互补的、 从根本上不同的方法将揭示这种生物本质反应的机制。 这些实验利用了包括合成有机化学、酶动力学、 生物化学和结构生物学。区域专一性，过渡态手性，退出路径 活性中心内的溶剂原子的同一性将使用 越来越复杂的活性部位抑制剂系列。底物辅助催化的本质 并通过测定P位的反应动力学来检验金属离子在底物活化中的作用 含有特定位点的化学替代的tRNA。化学过渡态的性质和化学过渡态 参与反应的每个原子上的键序和电荷的相对程度将由下式决定 动力学同位素效应分析。利用一系列具有广谱PKA的非天然氨基酸 值时，将测定肽基转移酶反应的Bronsted系数。这将提供 支持或反对一般碱或一般酸催化机制的进一步证据。酶通过以下途径发挥作用 与它们的过渡态结合得比它们的基态更紧密。确定电荷量的分布 过渡状态将使开发出针对核糖体的紧密结合的抗生素和 改进已用于治疗细菌感染的抗生素。 2-3句小结。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 将导致抗击疾病的抗生素的改进。

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.