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

Abstract

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 ( K i ) 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.

Temporal stimulus patterns drive differentiation of a synthetic coacervate

“…Such biomimetic ultrasensitive responses were recently enabled by coupling of enzymatic reactions with artificial photoresponsive inhibitors in a flow reactor, but are still rare in synthetic systems chemistry. 50 The present results demonstrate induction of differentiation of synthetic dipeptide-based coacervates driven by temporally distinct stimulus dynamics. The proposed system, which can decode dynamic (temporal) information, is applicable to a wide variety of synthetic stimulus-responsive supramolecules because it relies on a simple, general substrate competition mechanism.…”

Temporal Stimulus Patterns Drive Differentiation of a Synthetic Dipeptide-Based Coacervate

“…, assembly–disassembly). ,,,,,− Compared with these examples, one of the important aspects of our work is to combine the rate enhancement effect with competitive inhibition via continuous supply of diffusing O 2 , which establishes a unique system in response to temporally distinct stimulus patterns. Similar ultrasensitive responses were recently enabled by coupling of enzymatic reactions with artificial photoresponsive inhibitors in a flow reactor, but are still rare in synthetic systems chemistry …”

Rational Design of Functional and Tunable Nanozymatic Networks

“…A plethora of different strategies to reversibly and spatiotemporally control the activity of enzymes were developed in the past decades. 11,13,56,57 Surfactants occurred in any biological organism in the form of phospholipids as a main constituent of the cellular membrane. As providing charged interfaces and polar cores, surfactants were often adopted to mimic the microenvironment of bio-catalysis processes.…”

“…Indeed, natural DNA, RNA, and enzymes were used as building blocks to form dynamic networks triggered by auxiliary chemical or physical stimuli, leading to complex and programmed reaction pathways. These chemical reaction networks reveal adaptation, 9,10 signal propagation, 11 switching, 12 feedback, 13 intercommunication, 14 and clustering of networks. 15,16 The further development of CRNs suffers, however, from fundamental limitations: (i) Like nucleic acids and enzyme, natural matter lack enough availability for the large scales required for industrial applications, and are easy to be degraded and denatured in vitro, which impede their broad application as CRNs.…”