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. ? ? ?
| Hiller, David A; Zhong, Minghong; Singh, Vipender et al. (2010) Transition states of uncatalyzed hydrolysis and aminolysis reactions of a ribosomal P-site substrate determined by kinetic isotope effects. Biochemistry 49:3868-78 |
