Solvent Entropy Contributions to Catalytic Activity in Designed and Optimized Kemp Eliminases

Abstract

We analyze the role of solvation for enzymatic catalysis in two distinct, artificially designed Kemp Eliminases, KE07 and KE70, and mutated variants that were optimized by laboratory directed evolution. Using a spatially resolved analysis of hydration patterns, intermolecular vibrations, and local solvent entropies, we identify distinct classes of hydration water and follow their changes upon substrate binding and transition state formation for the designed KE07 and KE70 enzymes and their evolved variants. We observe that differences in hydration of the enzymatic systems are concentrated in the active site and undergo significant changes during substrate recruitment. For KE07, directed evolution reduces variations in the hydration of the polar catalytic center upon substrate binding, preserving strong protein-water interactions, while the evolved enzyme variant of KE70 features a more hydrophobic reaction center for which the expulsion of low-entropy water molecules upon substrate binding is substantially enhanced. While our analysis indicates a system-dependent role of solvation for the substrate binding process, we identify more subtle changes in solvation for the transition state formation, which are less affected by directed evolution.

Disassembling solvation free energies into local contributions—Toward a microscopic understanding of solvation processes

“…Resolving local solvent energies and entropies and disentangling solute–water and water–water contributions to both provides a new microscopic perspective, which enables detailed insights into the complex determinants of energy–entropy compensation mechanisms that represent one of the main challenges of model‐based predictions . While the examples presented in this article are restricted to small molecular solutes for clarity, each technique has been employed to larger biomolecular systems including proteins or have even been implemented into standard simulation packages . This allows for direct applications to simulations of relevant drug targets to improve rational drug‐design strategies, the characterization of biomolecular surfaces, or analysis of the heterogeneity of hydration water properties.…”

“…The minimum sampling rate for resolving these vibrations corresponds to 10-20 fs intervals, while previous implementations have used sampling rates as low as 8 fs. 21,52,53 In order to calculate the required time-correlation functions, a history of at least several hundred time frames is required, which eventually determines the frequency resolution of the VDOS that is central to the entropy evaluation. If the analysis is carried out as an a posteriori analysis of simulation trajectories, the required time resolution results in large disk space requirements for the storage of the trajectories that include coordinates and velocities.…”

“…The latter has been used to classify distinct species of hydration water molecules in the vicinity of small molecular solutes 21 as well as in the hydration shell of proteins. 52,53 6 | CONCLUSIONS In general, the information provided by the various methods described in this article provides vital insights into the interactions of molecular solutes with surrounding water. This information is crucial to formulate improved implicit solvent models, which are able to accurately describe solvent-mediated interactions beyond current solvent-accessible-surface area-based approximations that do not capture the impact of chemical context.…”

“…In case of rigid water molecules, the fastest vibrations are high‐frequency librations at roughly 1,000 cm −1 or 30 THz. The minimum sampling rate for resolving these vibrations corresponds to 10–20 fs intervals, while previous implementations have used sampling rates as low as 8 fs . In order to calculate the required time‐correlation functions, a history of at least several hundred time frames is required, which eventually determines the frequency resolution of the VDOS that is central to the entropy evaluation.…”

“…However, in addition to the thermodynamic information, the analysis implicitly provides detailed spatially resolved information on local dynamic properties of the solvent, such as short‐time diffusion (zero‐frequency response of the VDOS) and the spectrum of local vibrations. The latter has been used to classify distinct species of hydration water molecules in the vicinity of small molecular solutes as well as in the hydration shell of proteins …”