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Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations.

Research paper by David R DR Stevens, Sharon S Hammes-Schiffer

Indexed on: 23 Jun '18Published on: 23 Jun '18Published in: Journal of the American Chemical Society



Abstract

The enzyme human DNA polymerase η (Pol η) is critical for bypassing lesions during DNA replication. In addition to the two Mg2+ ions aligning the active site, experiments suggest that a third Mg2+ ion could play an essential catalytic role. Herein the role of this third metal ion is investigated with quantum mechanical/molecular mechanical (QM/MM) free energy simulations of the phosphoryl transfer reaction and a proposed self-activating proton transfer from the incoming nucleotide to the pyrophosphate leaving group. The simulations with only two metal ions in the active site support a sequential mechanism, with phosphoryl transfer followed by relatively fast proton transfer. The simulations with three metal ions in the active site suggest that the third metal ion may play a catalytic role through electrostatic interactions with the leaving group. These electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3'OH nucleophile, and also inhibit the subsequent proton transfer.