Origin of the Activity Drop with the E50D Variant of Catalytic Antibody 34E4 for Kemp Elimination

Abstract

In enzymes, multiple structural effects cooperatively lead to the high catalytic activity, while individually these effects can be small. The design of artificial enzymes requires the understanding and ability to manipulate such subtle effects. The 34E4 catalytic antibody, catalyzing Kemp elimination of 5-nitrobenzisoxazole, and its Glu50Asp (E50D) variant are the subject of the present investigation. This removal of only a methylene group yields an approximately 30-fold reduction in the rate for the catalyzed Kemp elimination. Here, the aim is to understand this difference in the catalytic performance. The mechanism of Kemp elimination catalyzed by 34E4 and the E50D mutant is elucidated using QM/MM Monte Carlo simulations and free energy perturbation theory. In both proteins, the reaction is shown to follow a single-step, concerted mechanism. In the mutant, the activation barrier rises by 2.4 kcal/mol, which corresponds to a 62-fold rate deceleration, in good agreement with the experimental data. The positions and functionality of the residues in the active site are monitored throughout the reaction. It is concluded that the looser contact with the base, shorter base-Asn58 contact, less favorable π-stacking with Trp91 in the transition state of the reaction, and different solvation pattern all contribute to the reduction of the reaction rate in the E50D variant of 34E4.

Computational strategies for the design of new enzymatic functions

“…71 The application of this strategy to the study of the mechanism of KE59, KE70 and several mutants rendered similar conclusions. 72 The study of the effect of the mutation Glu50Asp in the 34E4 catalytic antibody, showed an increase in the free energy barrier of 2.4 kcal·mol −1,73 in good agreement with experimental observation. Again, the same methodology allowed justifying the promiscuity of the 4B2 antibody in catalysing the allylic isomerization and the Kemp elimination reactions.…”

Challenges and Advances in Validating Enzyme Design Proposals: The Case of Kemp Eliminase Catalysis

“…Here it would be useful to consider several recent studies of the Kemp eliminase and related systems: the semiempirical MO-QM/MM study of Jorgensen and coworkers have provided reliable results for the water reference reactions (40), but the predicted trend in the protein (17) is not encouraging. More specifically, the MO-QM/MM approach performs nicely in exploring the effect of changing the distance between the donor and acceptor (i.e., the Glu to Asp mutation (41)). However, the real challenge is to reproduce the effect of changing the environment (which occurs in directed evolution experiments and is usually responsible for the catalytic activity) and this challenge has not been yet met by the current MO-QM/MM studies of Kemp eliminases (which drastically underestimated the barrier in the enzyme).…”

Engineering a model protein cavity to catalyze the Kemp elimination

“…A longstanding justification for enzyme design is that success demonstrates understanding of mechanism (11,17,33,34). As impressive as recent design efforts have been, the resulting enzymes have retained much of the complexity of the templates on which they were built, and have resisted full analysis of how structure encodes function (35).…”