Insights into the RNase A mechanism
EMSL Project ID
38205
Abstract
mRNA-polymers copied from DNA are degraded after transcription by cleavage of diphosphate bonds in the RNA backbone by Ribonuclease enzymes (RNase) as part of cell metabolism. In our study we employ high level DFT/B3LYP QM/MM reaction path optimization and free energy simulation methods implemented in the NWChem software package to investigate the cleavage mechanism of a key RNase, RNase A. This enzyme is an efficient catalyst for the hydrolytic cleavage of phosphate-diester linkages on the 3' side behind cytosine and uracil nucleotides. The catalytic process occurs in two steps, 1st an intramolecular transphosphorylation, where after a nucleophilic attack of the ribose O2' on the phosphorous of the phosphate diester backbone bond a cyclic phosphate and a protonated O5' terminated RNA fragment is formed. After this reaction the O5'H nucleotide leaves the catalytic pocket. In the second step the cyclic phosphate is hydrolyzed to form a final O3' phosphate terminated ribonucleotide. This enzyme has been the subject of extensive experimental and theoretical investigation for more than 50 years. Previously we studied and published simulation studies of the second step of the reaction. In our recent Grand-Challenge project (gc20904) work, we focuced on the 1st step of the reaction. There is no X-ray structure of the reactant state for the RNase A/RNA complex. Such a structure is necessary to initiate a reaction path calculation. We have been able to generate a RNase A/RNA complex candidate structure using the available X-ray structure of RNase A/DNA (single stranded) complex and simulation methods. After solvation, equilibration and QM/MM optimization of the system we successfully generated the cyclic phosphate product of the 1st reaction step and performed NEB (nudged elastic bend) calculations to investigate the reaction path. We have very promising results, however the transition state energy is higher than expected. Adding water molecules to the QM region decreases the reaction energy. However, we need to carry out some additional calculations to further validate this result. We are close to finishing this work but need additional computer time.
Project Details
Project type
Limited Scope
Start Date
2009-11-04
End Date
2009-12-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Elsasser BM, M Valiev, and JH Weare. 2009. "A Dianionic Phosphorane Intermediate and Transition States in an Associative AN+DN Mechanism for the RibonucleaseA Hydrolysis Reaction." Journal of the American Chemical Society 131(11):3869-3871. doi:10.1021/ja807940y