Directed evolution of KE59 required to introduce stabilizing mutations and resulted in 2000 fold increase in catalytic activity [22]. Optimization increased hydrophobicity Selleck Depsipeptide of the active site and raised the pKa of the catalytic base by desolvation. Orientation of the functional groups was adjusted by mutations at the rim, which affected active site geometry via changing dynamics [ 26]. An alternative rotamer of Trp-109 resulted in a stabilizing interaction with the general base, which contributed to improving activity. The HG-3 design was based on the catalytic antibody 34E4 and was optimized by a combination of crystallography

and MD [27•]. It employed an aspartate (D127) as the general base, aromatic residues to provide π-stacking for substrate interactions and polar residues (serine, threonine, glutamine) to donate a hydrogen bond to the isoxazolic oxygen of the 5-nitrobenzisoxazole. This Kemp eliminase design was evolved to the most efficient artificial catalyst, with kcat of 700 s−1, which provided 6 × 108 fold rate acceleration as compared to the uncatalyzed reaction [ 6••]. Activity of the HG3.17 variant originated in the extremely tight fit of the substrate, which was also enabled by a shortened hydrogen bond to the general base Asp127. It is often believed that tight packing, which was also observed in evolution of other designs [ 31 and 33], contributes to catalysis by

desolvating the substrate. Selleck PD0332991 In case of HG-3 however, similar pH profiles of the original

design and the evolved variant argue against medium effect. Hydrophobic contacts on the other hand can also optimize the arrangement of the functional groups and result in better preorganization. In the evolved HG3.17 Kemp eliminase the network of hydrogen- bonding interactions, which was enabled by the alternative substrate conformation, provided better stabilization GBA3 of the negatively charged TS. Although the original KE07 design was optimized for ground state desolvation, its laboratory evolution improved electrostatic preorganization around the TS [ 39 and 43]. To assess how this effect improves in enzyme evolution, reorganization energies of the original and the evolved KE07 variants were determined [ 28•]. Free energy profiles of the designed and the evolved KE07 variants were calculated by Free Energy Perturbation/Umbrella Sampling techniques resulting in activation barriers in good agreement with the experiments [37]. Although the reorganization energy of the KE07 design was less favorable than that of the corresponding reaction in water, it decreased significantly in directed evolution (by 27.4 kcal mol−1). Analyzing different contributions to the catalytic effect in the original and the evolved KE07 enzyme indicated that the reorganization energy was the most sensitive component of the catalytic effect, which was also amenable to optimization by directed evolution.