Mechanism of peptidyl transfer by the ribosome deduced by kinetic isotope effects

描述（由申请人提供）：核糖体是所有生物体中负责蛋白质合成的大型蛋白质-核酸复合物。自近50年前被发现以来，mrna定向合成蛋白质的机制一直是人们关注的焦点。虽然晶体结构和生化工作已经揭示了mRNA的解码，但核糖体中肽键形成的机制仍然未知。核糖体是细胞功能的核心组成部分，因此了解其机制至关重要。大核糖体亚基的晶体结构，肽键形成的位置，确定了活性位点的所有原子。然而，它不能明确地确定哪些原子是重要的，生化方法也没有定论。为了充分了解核糖体催化的机制，将确定化学步骤的过渡态。核糖体催化的肽键形成与相应的非催化反应之间的差异将表明核糖体如何提高反应速率。与结构和生化数据的比较将确定负责组。动力学同位素效应的测量将用于确定过渡态的结构。用较重的同位素取代一个原子会改变反应的速率，这与该原子在过渡态的相对键合是一致的。通过测量几个位点的动力学同位素效应，可以绘制过渡态结构并确定其在反应坐标上的位置。以这种方式确定的结构可以通过设计一个过渡态类似物来验证，它应该比不能准确重现过渡态的类似物更有效地抑制反应。这种方法将产生关于核糖体形成肽键的机制的宝贵信息，这是通过结构或生化方法无法获得的。这将是试图理解细胞重要过程的一个重要组成部分。

DESCRIPTION (provided by applicant): The ribosome is a large protein-nucleic acid complex responsible for protein synthesis in all organisms. Since its identification nearly fifty years ago, a great deal of attention has been focused on the mechanism for mRNA-directed protein synthesis. While crystal structures and biochemical work have revealed much about decoding of the mRNA, the mechanism of peptide bond formation in the ribosome is still unknown. The ribosome is a central component of cellular function, and therefore understanding its mechanism is of vital importance. The crystal structure of the large ribosomal subunit, the site of peptide bond formation, identified all the atoms in the active site. However, it cannot definitively identify which atoms are important, and biochemical approaches have been inconclusive. To fully understand the mechanism for catalysis by the ribosome, the transition state for the chemical step will be determined. Differences between ribosome-catalyzed peptide bond formation and the corresponding uncatalyzed reaction will indicate how the ribosome increases the reaction rate. Comparison with structural and biochemical data will identify the responsible groups. Measurement of kinetic isotope effects will be used to determine the structure of the transition state. Substitution of an atom with a heavier isotope changes the rate of a reaction in correspondence with the relative bonding of that atom in the transition state. By measuring kinetic isotopes effects at several sites, the transition state structure may be mapped and its location on the reaction coordinate established. The structure determined in this way may be validated by the design of a transition state analogue, which should inhibit the reaction more effectively than analogues which do not reproduce the transition state as accurately. This approach will yield invaluable information about the mechanism of peptide bond formation by the ribosome that is unattainable by structural or biochemical methods. It will be an important component of attempts to understand a vital process of the cell. Proteins carry out the vast majority of cellular processes, but it is an RNA enzyme, the ribosome, that makes all the proteins in the cell. Because the ribosome is essential for cell viability, it must be overexpressed in cancer cells; conversely, inhibition of ribosomes is lethal and therefore it is a common target of antibiotics. The mechanism of protein synthesis by the ribosome is a fundamental aspect of biology that is not yet fully understood.