(143b) Transition State Analogue-Based Computational Enzyme Design

Despite a handful of recent successes, computational design of active enzymes remains a difficult yet important task. Enzymes are ubiquitous commercially and achieve reaction rates that are typically unattainable with inorganic catalysts. Catalysts function by effectively lowering the reaction energy barrier, namely through transition state stabilization, ground state destabilization, or both. Because the transition state structure for a reaction is challenging to resolve, our design approach focuses on the use of transition state analogue compounds as proxies for the true transition state structure. The fundamental basis of catalysis (i.e., lowering the reaction energy barrier) in coordination with the use of transition state analogue compounds as proxies for the unknown transition state structure stimulates the derivation of correlating expressions that relate enzyme catalytic properties to computationally-accessible interaction energies.

Here, we will provide a detailed description of the new design approach predicated on the use of interaction energy calculations and transition state analogues. Furthermore, we will validate the correlating expressions using experimental data from a benchmark system and apply these expressions to alter enzyme specificity from a native substrate analogue towards a non-native substrate. The validation of the design technique for both the native substrate analogue and the non-native substrate provides the basis for a new computational enzyme design method- OptZyme. OptZyme is a novel design methodology that implements a structure-based design protocol for improving activity of an enzyme with a new substrate. We will show that the application of OptZyme results in a library of mutants with an improved catalytic property for a particular substrate. We will illustrate trends observed within the libraries of engineered enzymes and provide a structural basis for some of the common occurrences. Finally, we will analyze differences between the constructed libraries of mutants, with a special emphasis on point mutations that improve catalytic activity for a non-native substrate.