Local disorder is associated with enhanced catalysis in an engineered photoswitch.

The LOV2 domain is commonly harnessed as a source of light-based regulation in engineered optogenetic switches. In prior work, we used LOV2 to create a light-regulated Dihydrofolate Reductase (DHFR) enzyme and showed that structurally disperse mutations in DHFR were able to tune the allosteric response to light. However, it remained unclear how light allosterically activates DHFR, and how disperse mutations modulate the allosteric effect. A mechanistic understanding of these phenomena would improve our ability to rationally design new light-regulated enzymes. We used a combination of Eyring analysis and CD spectroscopy to quantify the relationship between allostery, catalytic activity, and global thermal stability. We found that the DHFR/LOV2 fusion was marginally stable at physiological temperatures. LOV2 photoactivation simultaneously: (1) thermally destabilized the fusion and (2) lowered the catalytic transition free energy of the lit state relative to the dark state. The energetic effect of light activation on the transition state free energy was composed of two opposing forces: a favorable reduction in the enthalpic transition state barrier offset by an entropic penalty. Allostery-tuning mutations in DHFR acted through this tradeoff, either accentuating the enthalpic benefit or minimizing the entropic penalty but never improving both. Many of the allostery tuning mutations showed a negative correlation between the light induced change in thermal stability and catalytic activity, suggesting an activity-stability tradeoff.