Recruiting mechanism and functional role of a third metal ion in the enzymatic activity of 5′ structure-specific nucleases

Enzymes of the 5′ structure-specific nuclease family are crucial for DNA repair, replication, and recombination. One such enzyme is the human exonuclease 1 (hExo1) metalloenzyme, which cleaves DNA strands, acting primarily as a processive 5′-3′ exonuclease and secondarily as a 5′-flap endonuclease. Recently, in crystallo reaction intermediates have elucidated how hExo1 exerts hydrolysis of DNA phosphodiester bonds. These hExo1 structures show a third metal ion intermittently bound close to the two-metal-ion active site, to which recessed ends or 5′-flap substrates bind. Evidence of this third ion has been observed in several nucleic-acid-processing metalloenzymes. However, there is still debate over what triggers the (un)binding of this transient third ion during catalysis and whether this ion has a catalytic function. Using extended molecular dynamics and enhanced sampling free-energy simulations, we observed that the carboxyl side chain of Glu89 (located along the arch motif in hExo1) flips frequently f...