| Popis: |
Lon is an ATP-dependent serine protease that degrades damaged, misfolded, and certain regulatory proteins in the cell in order to maintain proper cellular function. Lon exists as a homo-oligomer with one ATPase domain and one protease domain in each sub unit. Truncated ELon mutants were used to determine the functionality of the N-terminal domain. Through steady-state and pre-steady-state conditions it was found that despite the removal of ~30 % of the N-terminal, substrate degradation still takes place. A physiological substrate of Esherichia coli Lon (ELon) called lamda N (LN) was used to demonstrate that the directionality and rate of cleavage. Using fluorescent model substrates (dansylLN and sFRLN), as a tool, we have employed steady-state and pre-steady state kinetic techniques to evaluate the rate of translocation and cleavage under ATP conditions. The results obtained from these experiments suggest that ELon delivers the C-terminal of LN to the active site first followed by the N-terminal. The translocation of LN in the presence of ATP shows an increasing in step curve; this could be a result of changes in a conformation change of Lon as the fluorescent substrate moves closer to the active site. After complete translocation, cleavage of all peptide cleavage sites takes place within a short time frame. The initial burst of ATPase activity suggests it is correlated to the initial stage of the translocation step. This indicates that ATP hydrolysis occurs before complete substrate translocation. Collectively, these results can be taken and fit with a global explorer program to yield a possible mechanism for ELon. Using this information we can determine the differences it has with hLon (human Lon protease). Previously, ADP was found to inhibit ELon activity. However, ADP inhibition on hLon is unknown. The similarities and differences obtained from hLon can provide information in the development of new therapeutic approaches to cure certain bacterial diseases in human. |
