Enzymes are responsible for catalyzing a myriad of reactions necessary for life. Because enzymes play such an important role in human physiology, they are often targets for drugs and disease treatments. Naturally occurring enzymes are capable of catalyzing a wide variety of reactions, but imagine if we could design an enzyme to catalyze any reaction we wanted. We would be able to create new antibiotics easily to combat antibiotic resistance or to quickly synthesize chemicals for industrial applications. Scientists have made a lot of progress towards creating new enzymes, yet there are still roadblocks. Modifying existing enzymes through directed evolution is inefficient and limited by the need for high throughput screening methods. Conversely, in the case of rational design, we are missing key information for the technique to work at its full potential.
My research works to fill in the gaps in our knowledge to allow for the efficient development of new enzymes. A large portion of the scientific community focuses on determining the structure of enzymes and how the structure impacts function. While this work is enormously important, it doesn’t tell the full story. One major aspect that is often overlooked when examining structure-function relationships is that enzymes are dynamic molecules. This means that they physically move, bend, wiggle, and change shape during catalysis.
To study enzyme dynamics, I use temperature jump spectroscopy. There are only a few labs around the world that use this technique, and even fewer that use it to study enzymes. Temperature jump spectroscopy relies on rapidly initiating a change in equilibrium. For example, my samples contain enzymes and ligands. As determined by the equilibrium constant, some of the ligand is bound and some ligand is free in solution. The sample starts at equilibrium at a specified temperature. Then, a laser pulse is used to rapidly heat a small portion of the sample. The system must relax to a new equilibrium at the higher temperature. Since ligand binding is an exothermic reaction, there will be a net flux of ligands dissociating from the enzyme. However, as a system relaxes to a new equilibrium it will shift in the forward and reverse directions providing information about both processes. From this data I can determine the rate at which ligands are binding and unbinding, accompanying enzyme motions, and even conformational changes unrelated to ligand association. These changes occur on the microsecond timescale.
Although temperature jump spectroscopy could be applied to any number of enzymes, so far I’ve studied one enzyme in particular, dihydrofolate reductase (DHFR). It is a small ubiquitous enzyme that is well known for changing conformations during its catalytic cycle. Thus, it is a good starting place for understanding enzyme dynamics. Furthermore, DHFR is an important enzyme for nucleic acid synthesis. Since nucleic acid synthesis is necessary for cellular replication, DHFR inhibition is a strategy for anticancer and antibacterial agents.
Understanding the motions of DHFR could lead to the development of new inhibitors to combat resistance developed in certain cancers. The technique I use can be applied to other enzyme systems as well. By studying multiple enzymes we can build an understanding of enzyme motions in general, which can then be used to inform computational simulations for rational enzyme design. This would ultimately allow us to efficiently design new enzymes as well as new drugs.
Reddish, M. J.; Vaughn, M. B.; Fu, R.; Dyer, R. B. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase. Biochemistry 2016, 55 (10), 1485-1493.