A conserved asparagine in a ubiquitin‐conjugating enzyme positions the substrate for nucleophilic attack. Issue 22 (9th May 2019)

• Record Type: Journal Article
• Authors: Jones, Walker M.; Davis, Aaron G.; Wilson, R. Hunter; Elliott, Katherine L.; Sumner, Isaiah
• Is Part Of: Journal of computational chemistry. Volume 40:Issue 22(2019)
• Page Start: 1969
• Page End: 1977
• Publication Date: 2019-05-09
• Subjects: ubiquitin -- E2 -- Ubc13 -- ubiquitination -- molecular dynamics -- QM/MM -- BOMD; Chemistry -- Data processing -- Periodicals; 542.85
• DOI: 10.1002/jcc.25852
• Languages: English
• ISSNs: 0192-8651

Abstract

Abstract : 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. Abstract : Asparagine 79 in the ubiquitin‐conjugating enzyme, Ubc13, keeps the electrophile fixed and ready for nucleophilic attack. In mutated enzymes, the electrophile is no longer rigid, which impedes the progress of the reaction.