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A conserved asparagine in a ubiquitin‐conjugating enzyme positions the substrate for nucleophilic attack
Author(s) -
Jones Walker M.,
Davis Aaron G.,
Wilson R. Hunter,
Elliott Katherine L.,
Sumner Isaiah
Publication year - 2019
Publication title -
journal of computational chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.907
H-Index - 188
eISSN - 1096-987X
pISSN - 0192-8651
DOI - 10.1002/jcc.25852
Subject(s) - molecular dynamics , nucleophile , chemistry , molecular mechanics , asparagine , limiting , ubiquitin , tetrahedral carbonyl addition compound , computational chemistry , electrophile , enzyme , catalysis , biochemistry , mechanical engineering , engineering , gene
The mechanism used by the ubiquitin‐conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born–Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long‐held hypothesis and show that Ubc13‐catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step—the formation of a tetrahedral, zwitterionic intermediate—is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild‐type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.