Time-Resolved and Multi-Temperature Structural Biology to Understand Dynamic Enzyme Catalysis

Project Summary Enzymes, proteins which perform chemical reactions, are required for all of life. These proteins are amazing and specific catalysts that are tightly regulated to ensure proper cellular function. Dysregulation of enzymes is associated with many diseases, and inhibitors, which bind to and turn off enzymes, are often used as drugs to

modulate an enzyme’s activity and cure or manage diseases. Much has been learned about enzyme function and mechanism via structural biology, which informs researchers of the structure of the enzyme, and kinetics experiments, which yield information about how well the enzyme binds its substrate and how fast the reaction

proceeds. However, these experiments do not capture the structure of the enzyme as it performs its catalysis or give information about the changes in enzyme structure under different conditions. Such information would improve our understanding of how enzymes function, improving rational enzyme design outcomes that design

enzymes with new activities or other beneficial properties like improved stability or temperature adaptations, and improving inhibitor and drug design outcomes which would benefit from knowledge of the enzyme in different states. One of the goals of the proposed research is to combine enzyme structure and enzyme kinetics into one

experiment using time-resolved structural biology, which collects atomic resolution enzyme structure along the biochemical reaction pathway, uncovering reaction intermediates and structural fluctuations with millisecond time resolution. This work builds upon our previous work and method development by applying milli-second mix-and-

quench crystallography (MMQX) to new enzyme systems which are active drug targets due to their importance in metabolism. Specifically, we are applying our methods to amino acid catabolism enzymes, which break down amino acids, the building blocks of proteins, into other components to drive growth in the living cell. In addition,

we are also developing methods and studying these and other enzymes via multi-temperature crystallography, which perturbs the energy landscape of these enzymes and causes their structures to fluctuate as they find their equilibria at the new temperature. These multi-temperature experiments have previously been demonstrated to

observe loop opening and closing, cryptic and allosteric binding sites, and differential inhibitor binding to standard cryo-crystallography experiments. These research programs seek to better understand enzyme dynamics and catalysis, which will improve human health by increasing the success rate of future enzyme engineering and

inhibitor design studies.