Molecular dynamics simulations reveal a new role for a conserved active site asparagine in a ubiquitin-conjugating enzyme.

The role of a highly conserved active site asparagine (N79) in the ubiquitin conjugating enzyme, Ubc13, is probed using molecular dynamics simulations. Both wild type and mutant enzymes (N79A and N79D) are studied. Contrary to a popular hypothesis, we show that it is unlikely that N79 stabilizes a reaction intermediate, but instead preferentially hydrogen bonds to a loop near the active site. This keeps the sidechain carboxylate of an aspartate in the loop (D119) near the sidechain amine of the substrate lysine. Our simulations show that this distance increases in the mutants. D119 has been hypothesized to play a variety of roles in the enzyme, including deprotonating the substrate lysine, so changing this distance can have an effect on the enzyme's efficiency. Finally, we show that it is possible for the aspartate to deprotonate the substrate even across long distances if short water wires form that connect the proton donor and acceptor. Short water wires form with greater probability in the wild type than in mutant enzymes.