Zusammenfassung
Besides its capability to switch between two configurations, azobenzene converts light energy to vibrational energy (VE). Incorporation of the azobenzene-based unnatural amino acid AzoF can, hence, facilitate the transfer of VE into an enzyme. We were interested whether this VE transfer might increase conformational dynamics important for catalysis to ultimately promote enzyme activity. To this ...
Zusammenfassung
Besides its capability to switch between two configurations, azobenzene converts light energy to vibrational energy (VE). Incorporation of the azobenzene-based unnatural amino acid AzoF can, hence, facilitate the transfer of VE into an enzyme. We were interested whether this VE transfer might increase conformational dynamics important for catalysis to ultimately promote enzyme activity. To this end, allosteric enzymes are well-suited model systems since allosteric regulation is mediated by conformational dynamics. To promote catalysis via VE transfer, we selected two positions for incorporation of AzoF in the prominent allosteric bi-enzyme complex imidazole glycerol phosphate synthase (ImGPS). While W123 in the HisH subunit is close to the critical active site and a key player for allostery, S55 in the HisF subunit is more distant contributing only moderately to allostery, as shown via in-depth biophysical and computational evaluations. We further demon-strated that simultaneous irradiation of AzoF with 365 nm and 420 nm produces higher amounts of VE than monochromatic irradiation and results in the appearance of a third spectroscopic signal originating from the azobenzene moiety. Remarkably, enzyme activity increased upon dichromatic irradiation for ImGPS(W123AzoF) but not for wild-type ImGPS or ImGPS(S55AzoF) and returned to initial activity values in the dark indicating that the change in activity was caused by VE transfer. Our findings provide a proof-of-concept for promoting enzyme activity by VE transfer and open up further possibilities to study the relationship between dynamics and enzyme catalysis.
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