A conserved asparagine in a ubiquitin‐conjugating enzyme positions the substrate for nucleophilic attack

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.

Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler

“…In addition to entropic effects on the side-chain, the hydrocarbon linker length would also affect catalysis. For example, as shown for UBE2N (without UBE2V1-RING E3 partners), computational studies support a model where there is a precise “hole” fitting the lysine amine, and attack on the thioester carbonyl is rate-limiting 46 . Our data may suggest that the acceptor UB lysine itself is optimal not only for accessing the amine hole, but also for the chemistry of ubiquitylation.…”

On the possibility that bond strain is the mechanism of RING E3 activation in the E2-catalyzed ubiquitination reaction

“…3,20,26 Alternately, this asparagine has been hypothesized to stabilize an active site loop and to pre-organize the thioester. [30][31][32] Therefore, we examined how the open/closed Ubc13~Ub state affects the hydrogen bonding in the active site, and we examined if the presence of the E3 had an effect. Our results are shown in Figs.…”

“…In particular, we focus on the behavior of a highly conserved asparagine in the E2 that may stabilize a reaction intermediate, 3,20,[26][27][28][29] or may help preorganize the substrates. [30][31][32]…”

Twenty years of exceptional success: The molecular education and research consortium in undergraduate computational chemistry (MERCURY)

“…The faculty published 2 peer‐reviewed books, [ 238,239 ] 9 peer‐reviewed book chapters, [ 240–248 ] and 115 peer‐reviewed research papers. [ 249–363 ] This comes to 1.6 peer‐reviewed products/faculty/year during the 3‐year grant period, which is 3.2 times the rate of publication for natural science faculty at PUIs. [ 46 ]…”

“…[ 433–435 ] The group aims to determine and compare the reaction pathways for representative members of the acetyl and ubiquitin transfer family of enzymes using AIMD and QM/MM electronic structure calculations. [ 301,345 ] MERCURY resources are crucial to the success of this project.…”