Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in Out-of-Equilibrium Enzymatic Reactions

Ultrasensitivity is a ubiquitous emergent property of biochemical
reaction networks. The design and construction of synthetic reaction
networks exhibiting ultrasensitivity has been challenging, but would
greatly expand the potential properties of life-like materials. Herein,
we exploit a general and modular strategy to reversibly regulate the
activity of enzymes using light and show how ultrasensitivity arises in
simple out-of-equilibrium enzymatic systems upon incorporation of
reversible photoswitchable inhibitors (PIs). Utilizing a
chromophore/warhead strategy, PIs of the protease α-chymotrypsin were
synthesized, which led to the discovery of inhibitors with large
differences in inhibition constants (Ki) for the
different photoisomers. A microfluidic flow setup was used to study
enzymatic reactions under out-of-equilibrium conditions by continuous
addition and removal of reagents. Upon irradiation of the continuously
stirred tank reactor with different light pulse sequences, i.e., varying
the pulse duration or frequency of UV and blue light irradiation,
reversible switching between photoisomers resulted in ultrasensitive
responses in enzymatic activity as well as frequency filtering of input
signals. This general and modular strategy enables reversible and
tunable control over the kinetic rates of individual enzyme-catalyzed
reactions and makes a programmable linkage of enzymes to a wide range of
network topologies feasible.