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

描述(申请人提供)：核糖体是一种大型蛋白质-核酸复合体，负责所有生物体中蛋白质的合成。自从近50年前被发现以来，人们一直关注于信使核糖核酸诱导的蛋白质合成的机制。虽然晶体结构和生化研究已经揭示了许多关于mRNA的解码，但核糖体中肽键的形成机制仍不清楚。核糖体是细胞功能的中心组成部分，因此了解其机制至关重要。大的核糖体亚基的晶体结构，即多肽键的形成位置，确定了活性位置上的所有原子。然而，它不能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的催化机制，我们将确定化学步骤的过渡态。核糖体催化的多肽键形成和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度。与结构和生化数据的比较将确定责任群体。将使用动力学同位素效应的测量来确定过渡态的结构。用较重的同位素取代原子会改变反应的速度，与处于过渡态的原子的相对成键相对应。通过测量几个位置的动力学同位素效应，可以绘制过渡态结构图，并确定其在反应坐标上的位置。用这种方法确定的结构可以通过设计过渡态类似物来验证，该过渡态类似物应该比不能准确地再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多肽键的机制的宝贵信息，这是结构或生物化学方法无法获得的。这将是试图了解细胞的重要过程的重要组成部分。 蛋白质执行细胞的绝大多数过程，但构成细胞中所有蛋白质的是一种核糖体--核糖体--RNA酶。由于核糖体对细胞存活至关重要，它必须在癌细胞中过度表达；相反，抑制核糖体是致命的，因此它是抗生素的常见靶点。核糖体合成蛋白质的机制是生物学中尚未完全了解的一个基本方面。

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.