Electrostatic Preorganization\nin Three Distinct Heterogeneous\nProteasome β‑Subunits

The origin of the\nenzyme’s powerful role in accelerating\nchemical reactions is one of the most critical and still widely discussed\nquestions. It is already accepted that enzymes impose an electrostatic\nfield onto their substrates by adopting complex three-dimensional\nstructures; therefore, the preorganization of electric fields inside\nprotein active sites has been proposed as a crucial contributor to\ncatalytic mechanisms and rate constant enhancement. In this work,\nwe focus on three catalytically active β-subunits of 20S proteasomes\nwith low sequence identity (∼30%) whose active sites, although\nsituated in an electrostatically miscellaneous environment, catalyze\nthe same chemical reaction with similar catalytic efficiency. Our\nin silico experiments reproduce the experimentally observed equivalent\nreactivity of the three sites and show that obliteration of the electrostatic\npotential in all active sites would deprive the enzymes of their catalytic\npower by slowing down the chemical process by a factor of 10<sup>35</sup>. To regain enzymatic efficiency, besides catalytic Thr1 and Lys33\nresidues, the presence of aspartic acid in position 17 and an aqueous\nsolvent is required, proving that the electrostatic potential generated\nby the remaining residues is insignificant for catalysis. Moreover,\nit was found that the gradual decay of atomic charges on Asp17 strongly\ncorrelates with the enzyme’s catalytic rate deterioration as\nwell as with a change in the charge distributions due to introduced\nmutations. The computational procedure used and described here may\nhelp identify key residues for catalysis in other biomolecular systems\nand consequently may contribute to the process of designing enzyme-like\nsynthetic catalysts.