Toward accurate screening in computer-aided enzyme design

Abstract

The ability to design effective enzymes is one of the most fundamental challenges in biotechnology and in some respects in biochemistry. In fact, such ability would be one of the most convincing manifestations of a full understanding of the origin of enzyme catalysis. In this work, we explore the reliability of different simulation approaches, in terms of their ability to rank different possible active site constructs. This validation is done by comparing the ability of different approaches to evaluate the catalytic contributions of various residues in chorismate mutase. It is demonstrated that the empirical valence bond (EVB) model can serve as a practical yet accurate tool in the final stages of computer-aided enzyme design (CAED). Other approaches for fast screening are also examined and found to be less accurate and mainly useful for qualitative screening of ionized residues. It is pointed out that accurate ranking of different options for enzyme design cannot be accomplished by approaches that cannot capture the electrostatic preorganization effect. This is in particular true with regard to current design approaches that use gas phase or small cluster calculations and then estimate the interaction between the enzyme and the transition state (TS) model rather than the TS binding free energy or the relevant activation free energy. The ability of the EVB model to provide a tool for quantitative ranking in the final stage of CAED may help in progressing toward the design of enzymes whose catalytic power is closer to that of native enzymes than to that of the current generation of designer enzymes.

Figure 1

Rearrangement of chorismate to prephenate

Figure 2

Structures of the trimeric BsCM (left), dimeric EcCM (center) and monomeric mMjCM (right) proteins. The actives sites contain prepenate, TSA and TSA in the trimer, dimer and monomer, respectively. These active site ligands are represented by ball-and-stick models

Figure 3

(a) Schematic description of the EcCM and mMjCM active sites, depicting key residues that are involved in the binding of the transition-state analogue (bold). (b) Schematic description of the BsCM active site, depicting key residues that are involved in the binding of the transition-state analogue (bold). The targeted residues for simulations of mutational effects are colored red.

Figure 4

Relationship between the reaction barrier (Δg^‡) and the reorganization energy (λ) in aqueous solution and in an enzyme. In aqueous solution λ is large and Δg^‡ is large, whereas in enzyme environment λ and (Δg^‡) are small.

Figure 5

Electrostatic group contributions obtained by Eq. 11 for the TS binding in the native EcCM in kcal/mol. The contributions are for the residues of both subunits which are close to active site considered in our simulations.

Figure 6

Comparing the calculated (in blue) and observed (in red) activation energies for the indicated systems. The dash line designates the activation free energy of the reaction in aqueous solution.

Figure 7

Energy versus RMS from the correct structure for a case where we consider the rotamer library for the residues Asn84, Gln88 and Tyr91. Each number in RMS_torsion axis represents a 15 degrees range of torsional RMS. For example: 1 and 2 and 3 correspond to RMS of (–15) and (–30) and (–45), respectively. The energies reported correspond to the explicit model with implicit solvent rather that to the EVB free energies. The point is that the configurations with low energy are not far from the correct active site structure and thus can be used as reasonable starting points for the EVB calculations.