Molecular Gating of an Engineered Enzyme Captured in Real Time
Kokkonen, P., Sykora, J., Prokop, Z., Ghose, A., Bednar, D., Amaro, M., Beerens, K., Bidmanova, S., Slanska, M., Brezovsky, J., Damborsky, J., Hof, M.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 140: 17999–18008 (2018)
Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, a single-molecule fluorescence spectroscopy and molecular dynamics. Photoinduced electron-transfer – fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at single molecule level with the rate constants (kon = 1822 s-1, koff = 60 s-1) corresponding well with those from the pre-steady-state kinetics (k-1 = 1100 s-1, k1 = 20 s-1).