UC Merced

Understanding the relationship between enzyme structure and function is a central goal in structural biology, with important implications for enzyme design, drug discovery, and biotechnology. In this dissertation, we investigate the role of structural flexibility and dynamics in enzyme catalysis using engineered enzymes as model systems and advanced X-ray crystallography techniques.Our results demonstrate that optimal catalytic function arises from a balance between rigidity and flexibility. In relatively simple, single-step reactions, increased rigidity can enhance catalysis by preorganizing the active site to closely match the transition state geometry, thereby reducing the need for conformational rearrangement. In contrast, more complex, multi-step reactions require enzymes to sample multiple conformational states to accommodate substrates, intermediates, and products throughout the catalytic cycle. In these cases, conformational flexibility becomes essential for efficient function. Through structural and comparative analyses of evolved enzyme variants, we show how directed evolution modulates the conformational landscape of enzymes, enabling expansion or reshaping of the active site and selectively enriching catalytically productive states. Additionally, time-resolved and room-temperature crystallographic approaches provide insight into non-equilibrium conformations and transient states that are not accessible through conventional cryogenic methods. Together, these findings provide a unified framework for understanding how enzymes balance rigidity and flexibility to achieve efficient catalysis, and highlight the importance of dynamics in shaping enzyme function.