Mechanistic studies on substrate inhibition and substrate activation of ribonuclease A: experimental and in silico investigation

Ribonuclease A (RNase A) is an endonuclease that plays a significant role in antimicrobial activity by the cleavage and hydrolysis of ssRNA. In this study, the interactions between RNase A and cCMP have been investigated, through molecular docking and a 200 ns molecular dynamics simulation. The enzyme kinetic properties were analyzed using saturation curve, Eadie-Hofstee, and Hill plots. The docking results indicate that the cCMP-RNase A complexes are stabilized through hydrophobic interaction, hydrogen bonding, and & pi;-& pi; stacking interaction. The most binding site is observed in the catalytic cleft, especially at residue His12 and His119. Enzyme-ligand docking study indicates that cCMP binds to residues located in the internal cavity of the catalytic site and shows three phases of binding modes. The analysis of MD simulations shows that RMSD, Rg, and RMSF reach equilibrium. The & UDelta;G(binding) of the cCMP-RNase A was negative (-619.673 kJ/mol), The comparison between the results pre and post MD simulation showed that & UDelta;G(binding) after MD simulation causes to more stable the structure than before simulation. Experimental methods such as saturation, Eadie-Hofstee, and Hill plots confirm theoretical data and show three phases of binding modes as well. Two significant events are demonstrated in the interaction between RNase A and cCMP: substrate activation and substrate inhibition are observed in concentrations below and above 0.8 mM, respectively, for cCMP. Choosing the appropriate concentration of cCMP is very important in investigation of ribonuclease A's catalytic behavour, especially for exploration of the effects of some drugs on biological behaviours related to ribonuclease A.Communicated by Ramaswamy H. Sarma

Automated summary

This study investigates the interactions between RNase A and cCMP through molecular docking and molecular dynamics simulation. The results indicate that cCMP-RNase A complexes are stabilized through hydrophobic interaction, hydrogen bonding, and π-π stacking interaction. The enzyme-ligand docking study shows three phases of binding modes, and experimental methods confirm the activation and inhibition of substrate at different concentrations of cCMP.