Computational Enzyme Design: Refining Artificial Enzymes and Exploring Paths of Directed Evolution

Abstract

A fundamental challenge in biotechnology and in biochemistry is the ability to design effective enzymes. In fact, it would be one of the most convincing indications of a full understanding of the origin of enzyme catalysis. Despite a gradual progress on this front, most of the advances 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 (Chem. Rev. 2006, 106, 3210-3235). Improving the preorganization and assessing the effectiveness of different design options requires the ability to calculate the actual catalytic effect. Our studies are based on using the empirical valence bond (EVB) as the main screening tool (for e.g., Biochemistry 2009, 48, 3046-3056). At present this approach appears to provide the most reliable way for quantifying catalytic effects. Previously, we explored the challenges of the rational enzyme design in artificial Kemp eliminases with glutamate as a base (Frushicheva et al., PNAS, Sept 09 2010). It has been shown that due to the small change in substrate charges upon reaching the transition state makes it hard to exploit the active site polarity. This appears to be the case even with the ability to quantify the effect of different mutations. Thus we study the catalytic effect of the Kemp elimination reaction with different catalytic bases, such as histidine and lysine. This allowed us to exploit change in basicity as well the larger dipole of the transition charge distribution. We also investigate the effect of mutations that convert the highly catalytic phosphotriestrase to a lactonase. Overall we are able to illustrate the effectiveness of the EVB as a powerful approach for a quantitative screening in rational enzyme design.