The journal of physical chemistry. B 2017 10 04121(41) 9557-9565 doi 10.1021/acs.jpcb.7b08320
Enzymatic reactions usually occur in several steps: a step of substrate binding to the surface of the protein, a step of protein reorganization around the substrate and conduction of a chemical reaction, and a step of product release. The release of inorganic phosphate-PPi-from the matrix of the protein HIV reverse transcriptase is investigated computationally. Atomically detailed simulations with explicit solvent are analyzed to obtain the free energy profile, mean first passage time, and detailed molecular mechanisms of PPi escape. A challenge for the computations is of time scales. The experimental time scale of the process of interest is in milliseconds, and straightforward molecular dynamics simulations are in sub-microseconds. To overcome the time scale gap, we use the algorithm of Milestoning along a reaction coordinate to compute the overall free energy profile and rate. The methods of locally enhanced sampling and steered molecular dynamics determine plausible reaction coordinates. The observed molecular mechanism couples the transfer of the PPi to positively charged lysine side chains that are found on the exit pathway and to an exiting magnesium ion. In accord with experimental findings, the release rate is comparable to the chemical step, allowing for variations in substrate (DNA or RNA template) in which the release becomes rate determining.