Exploring challenges in rational enzyme design by simulating the catalysis in artificial kemp eliminase

Abstract

One of the fundamental challenges in biotechnology and in biochemistry is the ability to design effective enzymes. Doing so would be a convincing manifestation of a full understanding of the origin of enzyme catalysis. Despite an impressive progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the environment preorganization, which is the key factor in enzyme catalysis. Rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work takes previously designed kemp eliminases as a benchmark for a computer aided enzyme design, using the empirical valence bond as the main screening tool. The observed absolute catalytic effect and the effect of directed evolution are reproduced and analyzed (assuming that the substrate is in the designed site). It is found that, in the case of kemp eliminases, the transition state charge distribution makes it hard to exploit the active site polarity, even with the ability to quantify the effect of different mutations. Unexpectedly, it is found that the directed evolution mutants lead to the reduction of solvation of the reactant state by water molecules rather that to the more common mode of transition state stabilization used by naturally evolved enzymes. Finally it is pointed out that our difficulties in improving Kemp eliminase are not due to overlooking exotic effect, but to the challenge in designing a preorganized environment that would exploit the small change it charge distribution during the formation of the transition state.

Fig. 1.

A schematic description of the kemp elimination reaction.

Fig. 2.

The structure of the native enzyme (PDB: 2RKX) with the docked substrate.

Fig. 3.

Correlation between the calculated and observed activation barriers.

Fig. 4.

The group contributions (in kcal/mol) that reflect the interactions between the protein residues with the charge change upon moving from the RS to the TS (the ΔQs in Eq. 1). The calculations are done with ε_ij = 4 for all residues. The largest negative contributions provide a rough guide for the optimal sites for effective mutations that would enhance the catalytic effect.

Fig. 5.

The electrostatic potential form the change of the substrate charges upon moving from the RS to the TS (red and blue for negative and positive potential respectively) and group contributions (in kcal/mol) of key residues, where the contributions are defined for the case where each residue is positively charged.

Fig. 6.

Showing the water penetration near Glu101 in the wt and the R6 3/7 F mutant. As seen from the figure there are fewer water molecules near Glu101 in the mutant.