The Importance of the Scaffold for de Novo Enzymes: A Case Study with Kemp Eliminase

Abstract

We report electric field values relevant to the reactant and transition states of designed Kemp eliminases KE07 and KE70 and their improved variants from laboratory directed evolution (LDE), using atomistic simulations with the AMOEBA polarizable force field. We find that the catalytic base residue contributes the most to the electric field stabilization of the transition state of the LDE variants of the KE07 and KE70 enzymes, whereas the electric fields of the remainder of the enzyme and solvent disfavor the catalytic reaction in both cases. By contrast, we show that the electrostatic environment plays a large and stabilizing role for the naturally occurring enzyme ketosteroid isomerase (KSI). These results suggest that LDE is ultimately a limited strategy for improving de novo enzymes since it is largely restricted to optimization of chemical positioning in the active site, thus yielding a ∼3 order magnitude improvement over the uncatalyzed reaction, which we suggest may be an absolute upper bound estimate based on LDE applied to comparable de novo Kemp eliminases and other enzymes like KSI. Instead de novo enzymatic reactions could more productively benefit from optimization of the electrostatics of the protein scaffold in early stages of the computational design, utilizing electric field optimization as guidance.

Computational Optimization of Electric Fields for Improving Catalysis of a Designed Kemp Eliminase

“…We only give a brief description here, and more specific details for each of the simulation techniques have been provided in a previous publication 34 .…”

“…We then performed 50 ps equilibration and then 50 ps production runs in the NPT ensemble using the Tinker software package [49][50][51] and the AMOEBA polarizable force field 45,49 to provide a high quality description of electrostatics in the active site and overall scaffold and solvent. The substrate geometry for the transition state The electric field is projected onto each bond and the value reported is given by the mean of the electric field at the two atoms involved 34 . …”

“…al 17 ) and stabilization of the transition state (EL † ) to enhance activity, with significant contributions from side chain entropy that supported this trend 36 . In the second study, we demonstrated how electric field stabilization of the three bonds of the substrate that are broken and formed to create the product was greatly improved in the evolved KE07 enzyme, while for KE70 the LDE strategy evolved toward mutations that created a more hydrophobic active site instead 34 . In our study on KE07 and KE70, a breakdown of contribution of the electric field from individual residues further revealed that chemical positioning of the catalytic base and active site residues in the immediate vicinity of the substrate were the primary source of electrostatic improvement for both enzymes.…”

“…Finally, Warshel and coworkers were the first to identify the preorganization of the electrostatic free energy as a key component of enzyme catalysis [26][27][28] ; in fact many groups have reported evidence for how electric fields contribute to the catalytic power of various natural enzymes. [29][30][31][32][33] (a) (b) (c) We have recently reported in two separate studies the effects of both side chain conformational entropy and mutual information 36 , as well as electrostatic stabilization 34 , for improving designed KE07 and KE70 enzymes during the LDE process. It was shown in the first study that improved variants used a combination of free energy destabilization of the reactant enzyme-ligand bound state (EL) (in agreement with Frushicheva et.…”

“…In our study on KE07 and KE70, a breakdown of contribution of the electric field from individual residues further revealed that chemical positioning of the catalytic base and active site residues in the immediate vicinity of the substrate were the primary source of electrostatic improvement for both enzymes. In contrast, the solvent and the remaining scaffold provided electrostatic environments that were detrimental to the active site chemistry for KE07 and KE70, unlike native enzymes 34 .…”