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

Article abstract of DOI:10.1021/jacs.0c12956

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.

Full text of DOI:10.1021/jacs.0c12956

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Article
Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in
Out-of-Equilibrium Enzymatic Reactions
Michael Teders, Aleksandr A. Pogodaev, Glenn Bojanov, and Wilhelm T. S. Huck*
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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 (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.
systems remains challenging due to the lack of a general strategy
enabling the reversible and tunable regulation of enzymes under
out-of-equilibrium conditions.
INTRODUCTION
■
Living systems display unique capabilities, e.g., adaptation to the
environment, self-healing, homeostasis, or converting chemical
energy into directed motion, growth, and division. These
processes are governed by complex chemical reaction networks
that operate far from equilibrium and allow a precise regulation
of a wide range of cellular mechanisms, e.g., signaling or
metabolism.1−3 A characteristic feature found in many
biochemical networks is ultrasensitivity, which means that (in
contrast to a standard hyperbolic Michaelis−Menten response)
the response to a stimulus yields a sharp, switch-like sigmoidal
function (see Figure 1A for a general schematic of the
phenomenon).4−7 This property enables signaling systems to
filter out noise and be readily activated once a certain required
threshold stimuli is present. Different mechanisms have been
identified that can generate this nonlinear input−output
relationship, e.g., multisite phosphorylations,6 molecular titra-
tions (buffering),8 substrate competition,9,10 or zero-order
kinetics.4
Although a plethora of different external stimuli to reversibly
and spatiotemporally control the activity of enzymes have been
applied in the last decades,23 light is an ideal external control
element: it is bioorthogonal (λ > 360 nm),24−27 offers high
spatiotemporal resolution, can be precisely tuned in terms of
photon flux, and introduces the opportunity to control chemical
reaction networks using optoelectronic devices.28−30 A prom-
inent approach to gain photocontrol over diverse biological
processes is the (mostly) covalent installation of photo-
switchable chromophores into the biomolecule of interest.31−35
These so-called “molecular photoswitches” undergo a reversible
change in their three-dimensional structure between two or
more isomeric forms upon irradiation with light of suitable
wavelengths. A number of biological processes, such as protein
folding,36 membrane transport,37 or transcription and trans-
A central goal of systems chemistry is to investigate and
translate the common design principles of nature into a practical
and modular approach, ultimately enabling a programmable and
rational design of life-inspired systems exhibiting tunable
properties.11−18 While (light-induced) sigmoidal responses in
enzymatic logic gate systems have been reported,19−22 the
bottom-up construction of ultrasensitive, life-inspired enzymatic
Received: December 14, 2020
Published: April 12, 2021
© 2021 The Authors. Published by
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systems, the photoswitchable inhibitors need to fulfill several
criteria:
(1) The switching factor (SF), i.e., the ratio between the
inhibition constants of the cis and trans isomer
(SF = Ki,cis/Ki,trans), should be bigger than 10. This ensures
that the activity of the respective enzyme can be
sufficiently externally modulated using light.
(2) The PIs should exhibit high photostationary states (PSS,
defined as the percentage of cis isomer present at
equilibrium under irradiation, PSS = ([cis-PI]/([cis-PI ]
+ [trans-PI]) × 100) under UV (365 nm) irradiation. A
high PSS (>90%) implies superior concentrations of the
cis isomer relative to its trans isomer, thereby enabling
decent levels of photocontrol over the activity of the
respective enzyme.
(3) In order to reversibly regulate the activity of enzymes
using light, the PI needs to exhibit robust photo-
reversibility. This implies that no observable decom-
position of the PI should take place upon several
consecutive UV/vis switching cycles (>10).
(4) Ideally, the inhibition constants (Ki) of both the trans and
cis isomer of the PI should be in the μM range or lower.
Thereby, only small quantities of PIs, enzymes, and
substrates have to be used throughout the performance of
flow experiments.
Figure 1. (A) Schematic of Michaelis−Menten (black) and ultra-
sensitive response (blue). (B) Incorporation of competitive photo-
switchable inhibitors into out-of-equilibrium enzymatic systems
enables reversible and tunable activity control using light.
Enz = enzyme, W = warhead.
(5) Additionally, the PIs have to be sufficiently water-soluble,
should exhibit high photostability under irradiation, and
should show slow thermal cis to trans isomerization, as
complete photocontrol over the state of the PIs using light
is preferred.
lation,38 have successfully been photoregulated by the
incorporation of photosensitizable units. Thus, an attractive
option for obtaining optical control over enzyme function is by
installation of molecular photoswitches into the scaffold of
enzyme inhibitors.23,34,39
Diverse photoswitchable inhibitors for the photoregulation of
the activity of proteases have been described in the
literature.43−47 These effectors were mostly rationally designed
by incorporating a molecular photoswitch (usually an
azobenzene) into a previously identified pharmacophore (a
reversible inhibitor functionality; “warhead”), thus applying a
so-called chromophore/warhead strategy.34 However, the
reported PIs exhibit suboptimal properties for successful
application in out-of-equilibrium enzymatic systems due to
several reasons, e.g., the best Cr PI, bearing a trifluoromethyl
ketone warhead, had a SF of only 4.7.47
Therefore, we set out to design and synthesize improved
photoswitchable Cr inhibitors to fulfill these criteria and render
them suitable for application in out-of-equilibrium enzymatic
systems. The synthesis of PIs was performed by applying a
chromophore/warhead strategy,34 aiming to maximize the
difference in Ki between both isomeric states (switching factor),
while keeping the other (photo-)physical requirements for later
flow applications in mind. Due to their excellent photophysical
properties and straightforward synthetic construction, azoben-
zenes were chosen for the chromophore.48 Based on initial
experiments, phenylalanine-, tyrosine-, valine-, tryptophan-,
boronate-, or boronic-acid-based warheads were identified as
unsuitable when installed into the azobenzene scaffold. Weak
inhibitory properties, negligible differences in Ki between both
isomeric states, and insufficient water solubility or (photo-
chemical) degradation of the respective PIs over time were
observed. Inspired by a report from Smoum et al. on the
competitive inhibition of α-chymotrypsin by trifluoro(organo)-
borates,49 we aimed to synthesize azobenzene-based photo-
switchable inhibitors bearing this warhead. A systematic
structure−activity-relationship (SAR) analysis was performed,
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). We studied enzymatic activity of a α-
chymotrpysin/PI-based system under out-of-equilibrium con-
ditions (continuous feeding of substrates and waste removal)
using a continuously stirred tank reactor (CSTR) and
investigated the response to a series of light pulses (UV (365
nm) and blue (460 nm) light) as input stimuli (Figure 1B).
Surprisingly, we discovered a very strong ultrasensitive response
to combinations of UV and blue light pulses under out-of-
equilibrium conditions. This nonlinear response was also
observed in a bottom-up constructed enzymatic cascade based
on the proteolytic activity of α-chymotrypsin (Cr) and
aminopeptidase M (Ap). Furthermore, we observed that a
combination of flow and reversible activity regulation yields a
simple enzymatic system with frequency filtering properties
upon gradually increasing the UV pulse irradiation frequency
and switching back the PI using blue light.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Competitive,
Photoswitchable Inhibitors of α-Chymotrypsin (PIs).
Due to our long-standing expertise in the bottom-up
construction of enzymatic reaction networks using pro-
teases,29,40−42 we started by synthesizing reversible photo-
switchable inhibitors of α-chymotrypsin (PIs) in order to
externally control its enzymatic activity using light. To
sufficiently regulate the output of out-of-equilibrium enzymatic
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in which the position of the BF3K warhead on the azobenzene
chromophore was altered and diverse substituents with different
steric and electronic properties were introduced (see 2.1−2.5 of
the Supporting Information (SI) for details). In total, 21
photoswitchable α-chymotrypsin inhibitors have been synthe-
sized throughout the performed SAR study. The synthesized PIs
have been carefully characterized with respect to (photo-
)physical and inhibitory properties (see 3.1−4.6 of the SI for
detailed results). Among them, we identified α-chymotrypsin
inhibitor PI, bearing a meta-trifluoroborate functionality, as an
ideal candidate for our envisaged applications (Figure 2A, see
2.1−2.5 of the SI for details).
using a fluorogenic assay.42 To our delight, trans-PI was
identified to be a (moderately) strong competitive inhibitor of
α-chymotrypsin (Ki,trans = 12.6
0.6 μM), whereas cis-PI
exhibited significantly weaker inhibitory properties
(Ki,cis = 264.2 50.6 μM). The three-dimensional structural
change caused by photoinduced trans to cis isomerization thus
leads to weaker interactions of cis-PI and Cr compared to trans-
PI. Combined, this leads to a good switching factor of 21.0 and
enables excellent reversible photoregulation of Cr activity.
Having identified PI as a suitable Cr inhibitor, we
experimentally probed and verified its reversible photoswitching
properties upon consecutive 365/460 nm irradiation cycles as
well as its competitive mode of inhibition. We performed a batch
photoswitching experiment, in which a mixture of PI and α-
chymotrypsin in a TRIS/Ca2+/BSA (200 mM, 20 mM, 0.1 wt %,
pH = 7.81) buffer was successively irradiated several times for 5
min with light of a 365 or 460 nm LED, followed by the addition
of AAPF-AMC as substrate to an aliquot of the mixture (see 4.5
of the SI for detailed procedure and results). As depicted in
Figure 2B, the degree of AMC production over time was
reproducible throughout multiple 365/460 nm photoswitching
cycles, thereby demonstrating the reversible switching proper-
ties of PI as well as its competitive mode of α-chymotrypsin
inhibition. In addition, these results point toward high
photostability of Cr with respect to irradiation, as its
performance is still consistent and reproducible after a total of
40 min of each 365 and 460 nm irradiation.
Multi-State Activity Control over Cr in Enzymatic
Systems. With suitable PIs in hand, we aimed to regulate the
activity of the protease Cr in a reversible and tunable manner
under out-of-equilibrium conditions (continuous addition and
removal of PI, enzyme, and buffer to/from CSTR) using 365/
460 nm irradiation pulses. To this end, a microfluidic flow setup
with a CSTR was constructed. The latter could be selectively
irradiated with light from surrounding 365 and 460 nm LEDs,
which in turn were externally controlled by connecting both to
an Arduino device. The construction of this setup enabled a
programmable timing of the irradiation source and its intensity
(photon flux), the precise control over the flow rates of the
respective stock solutions, and on-line fluorescence monitoring
(Figure 3A; see 5.1 and 6 of the SI for experimental details of the
flow setup and computer code to control irradiation patterns).
To demonstrate multi-state activity control over Cr in this
Figure 2. (A) Structure and enzymological data of the photoswitchable
α-chymotrypsin inhibitor PI identified throughout structure−activity-
relationship analysis. Inhibition constants were determined using a
fluorogenic assay. For details on other synthesized photoswitchable α-
chymotrypsin inhibitors, experimental procedures, and results, see 2.1−
4.6 of the SI. SF = switching factor; PSS = photostationary state. (B)
Batch photoswitching using PI illustrating reversible photoswitching
and competitive mode of inhibition. [Cr] = 10 nM, [PI] = 400 μM,
[AAPF-AMC] = 80 μM (see 4.5 of the SI for details).
setup, we used PI (Ki , t r a ns = 12.6
0.6 μM;
Ki,cis = 264.2 50.6 μM) and AAPF-AMC as fluorogenic
substrate (Figure 3B). Irradiation with 365 nm light converts the
thermally adapted trans-PI into the weaker inhibitory cis isomer,
which induces an increase in Cr activity. By consecutive
irradiation with 460 nm light, the cis isomer of PI is converted
back to the stronger inhibitory trans isomer, leading to a
decrease in Cr activity. The amount of cis-PI present at the 365
nm photostationary state depends strongly on the applied
photon flux,48 and variation in light intensity therefore
introduces an easy route to modulate the effective inhibitor
strength. Thus, similar to the recently described light engineer-
ing of the PSS for wavelength-gated adaptation of hydrogel
properties,50 a wide range of enzymatic activity can be accessed
by utilizing PIs, which enables fine-tuning the kinetics of
enzyme-catalyzed reactions using light. Figure 3C shows how
irradiation with 365 and 460 nm light is translated into a well-
controlled temporal pattern of Cr activity. By applying the
depicted light pulse sequence, which is characterized by changes
in the photon flux of the 365 nm light source and differently long
PI exhibits a good solubility in commonly used aqueous
protease buffers (TRIS (200 mM), Ca2+ (20 mM), pH = 7.81),
and no substantial hydrolysis of the trifluoroborate was observed
within 24 h using 19F NMR analysis. In addition, the
photophysical characterization revealed both a high photo-
stability and photoreversibility of PI upon 365/460 nm
excitation and a very slow thermal cis to trans relaxation
(t1/2 = 274 h), thereby enabling full photocontrol over the
isomeric state of PI using 365/460 nm light. Importantly, the
content of cis isomer obtained upon 365 nm irradiation of PI in
the protease buffer was sufficiently high (PSS = 93%, see 3.1−3.4
of the SI for details regarding photophysical characterization of
PI).
Using PI, the protease α-chymotrypsin, N-succinyl-Ala-Ala-
Pro-Phe-7-amido-4-methylcoumarin (AAPF-AMC) as sub-
strate and TRIS/Ca2+/BSA as buffer (200 mM, 20 mM,
0.1 wt %, pH = 7.81), we determined the inhibition constants
(Ki) for the enzyme/inhibitor interaction of trans/cis-PI and Cr
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Inspired by these biological processes, we first systematically
altered the duration of irradiation with 365 nm light as input
stimuli, both with and without irradiation by blue light
immediately afterwards, in order to optionally switch the
chromophore back (Figure 4A). Due to the very slow thermal cis
to trans isomerization of PI (t1/2 = 274 h), the isomeric state of
the photoswitchable inhibitor is controlled exclusively by 365
and 460 nm irradiation. Thus, PI remains in its cis state after
365 nm irradiation and can only be converted back to the
corresponding trans state by 460 nm irradiation. In both pulse
sequences, the duration of UV irradiation (365 nm) was
gradually increased from 1 s to 30 min. The CSTR was irradiated
with the next UV pulse only after the AMC concentration had
stabilized to the initial concentration prior to UV irradiation. In
the absence of blue light irradiation, PI cannot undergo
photoinduced cis to trans isomerization. Hence, the AMC
output of the system to values prior to irradiation can only be
recovered “passively” by relying on flow to flush the cis isomer
out of the CSTR over time. In the pulse sequence with blue light
irradiation, however, a 15 s long pulse of blue light directly after
UV irradiation induces complete cis to trans photoisomerization,
thereby directly resetting the system to the Cr activity state prior
to UV irradiation. Figure 4A shows the enzymatic systems
response to the two pulse sequences, where the UV pulse
duration is logarithmically plotted against the corresponding
normalized AMC output recorded on-line using our flow setup.
In the absence of a blue light pulse, a sigmoidal response curve
was observed in which higher concentrations of AMC are
formed with increasing UV irradiation times. Just 5 s of UV
irradiation led to an increase in AMC output. The maximum Cr
activity of the enzymatic system was reached at UV irradiation
times of 5 min and longer. By irradiating the CSTR with a 15 s
long pulse of blue light immediately after each UV irradiation
step, a significantly different AMC response curve was obtained.
Only UV pulses of 3 min or longer led to an increase of AMC
production. Shorter UV pulses are filtered out, as the
combination of flow and the 460 nm induced cis to trans
isomerization results in a quick recovery of the system to the Cr
activity prior to UV irradiation. In both pulse sequences, we
observed an ultrasensitive input−output relationship, but
especially pulsing with blue light resulted in sharp, switch-like
behavior.5 This ultrasensitive response is caused by the interplay
of residence time of the Cr/PI mixture within the CSTR and the
duration and intensity of UV/blue light pulses. We wish to
remind the reader that an ultrasensitive response formally refers
to an increase from 10% to 90% of the maximum response upon
a less than 81-fold change in input stimulus. We can quantify the
degree of ultrasensitivity by fitting our experimental data to the
Hill equation (eq 1):
Figure 3. (A) Schematic of the experimental setup developed for the
performance of flow experiments. (B) Network motif of the Cr-
catalyzed cleavage of AAPF-AMC to give AMC. (C) Precise reversible
photocontrol over the activity of α-chymotrypsin under out-of-
equilibrium conditions: generation of an electrocardiogram heartbeat
AMC-output pattern. See 5.2.1 of the SI for details regarding timing,
duration, and intensity of 365 and 460 nm irradiation. Concentrations
in the flow cuvette: [Cr] = 40 nM, [PI] = 200 μM, [AAPF-AMC] =
80 μM. Flow rates: Cr, PI = 400 μL/h; AAPF-AMC, buffer = 200 μL/h.
Reactor volume = 196 μL.
pulses of 365 and 460 nm irradiation with 100% power, a
characteristic pattern could be introduced (see 5.2.1 of the SI for
details regarding timing, duration, and intensity of 365 and 460
nm irradiation). Interestingly, it appears that irradiation with
blue light (460 nm) can sharpen the peak generated by
irradiation with 365 nm (see large peak in the center). The
photoinduced trans to cis isomerization of PI is rapidly reversed
upon 460 nm irradiation, leading to the formation of a stronger
inhibitor and thus to a sharper decrease in Cr activity than
observed when the inhibitor concentration decreases only due
to efflux from the reactor (see smaller peaks).
Stimulusn
Response =
(ECn50 + Stimulusn)
(1)
Ultrasensitivity in Out-of-Equilibrium Enzymatic Sys-
tems by Incorporation of PIs. Encouraged by the initial
results shown above, we decided to study the impact of varying
the dynamics of the UV input. In cellular signaling pathways,
information is encoded not only in the amplitude of a certain
signal, but also in its dynamics.51,52 Hence, cellular output
decisions can be altered by differences in signal duration and
frequency of the input.53 It has been reported that changes to
dynamic signal transduction properties, e.g., by cancer
mutations or drugs targeting the respective pathway, can lead
to improper cell decisions and dysfunction.54−56
where ECn50 is the stimulus value needed to produce 50% of the
maximum response. This plotting allowed us to determine the
Hill coefficient (n), which is a quantitative measure of an
ultrasensitive response as defined according to eq 2:57
log[81]
n =
Ä
Å
Å
É
Ñ
Ñ
EC
EC
90
Å
logÅ
Å
Ñ
Ñ
Ñ
Ñ
Å
(2)
10
Å
Ç
Ñ
Ö
Herein, EC90 and EC10 are the stimulus values needed to
produce 90% and 10% of the maximum response, respectively.
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In this equation, a Michaelis−Menten response is characterized
by a Hill coefficient of n = 1. In signal transduction, the Hill
coefficients of ultrasensitive signaling systems are typically in the
range n = 1.3−20,5 for example with n = 3.5 for the Cdk1/
Wee1A stimulus−response relationship or n = 11 for Cdk1/
Cdc25C.9,58
In the absence of blue light pulses, the ultrasensitivity of the
enzymatic system is weak, with n = 1.25. In contrast, a highly
ultrasensitive response is obtained when UV and blue light
pulses are combined (n = 5.71), due to 460 nm induced cis to
trans isomerization causing a quick recovery of the system to the
Cr activity prior to UV irradiation.
To further rationalize and support our experimental findings,
we simulated the system’s response to the different pulse
sequences and UV pulse durations using mass-action kinetics
(Figure 4B). The model qualitatively reproduces the sigmoidal,
switch-like behavior observed experimentally very well. We note
that there is a global shift to shorter UV pulse durations leading
to a faster response in the simulations compared to the
experimental data. This discrepancy is caused by the fact that we
only have rough estimates of the rates of photoswitching (for
details, see 7 of the SI). The exact position of the plateau is
determined by both trans to cis and cis to trans photo-
isomerization rates as well as by the applied UV pulse durations.
To illustrate how our regulatory strategy can be applied to
control more complex systems, we designed an enzymatic
cascade that consists of two elemental steps. The first one is the
Cr-catalyzed proteolytic cleavage of the phenylalanine/arginine
peptide bond (KM = 180 μM) of the substrate Z-Phe-Arg-AMC,
which produces H-Arg-AMC (Figure 4C).41 The peptide bond
of the latter molecule is then cleaved by the action of
aminopeptidase M (Ap),59 thereby producing the AMC
reporter molecule (KM = 5 mM). As the AMC production of
the cascadic network depends on the Cr-catalyzed formation of
H-Arg-AMC, the whole output of the network can be controlled
via photoinduced isomerization of PI. Similar to the experiments
performed before, we systematically altered the duration of
irradiation with 365 nm and optionally switched the
chromophore back with blue light irradiation immediately
afterwards. Regardless of the applied pulse sequence, both
curves show an ultrasensitive response (Figure 4C). Interest-
ingly, in the absence of blue light pulses, we determined a Hill
coefficient of n = 1.46, which correlates with stronger
ultrasensitivity for the Cr/Ap-based system compared to the
single enzyme Cr/PI-based system discussed above (n = 1.25).
In the two-step cascade, short UV pulses do not lead to the
formation of significant amounts of H-Arg-AMC. As also the Ap-
catalyzed cleavage is slow due to weak binding of the substrate
(KM = 5 mM), no substantially increased amounts of AMC can
be formed before the enzyme/substrate mixture reaches the
fluorescence detector unit. Thus, even in the absence of a blue
light pulse, the enzymatic system is able to filter short UV pulses
due to the kinetics of the two underlying elemental steps. By
irradiating the CSTR with a 10 s long pulse of blue light after
each UV-irradiation step, the system became more ultrasensitive
compared to the other pulse sequence (n = 2.78); however, this
ultrasensitivity is significantly less pronounced compared to that
of the simpler Cr/PI-based system (n = 5.71). The lowered
ultrasensitivity is a result of the irreversible production of H-Arg-
AMC, which will proceed to AMC production, even when Cr is
inhibited again after the blue light pulse. These results provide a
proof-of-concept demonstration of our strategy to induce and
control the ultrasensitivity in enzymatic cascades and to
Figure 4. (A) Impact of different UV pulse durations and pulse
sequences (with and without 15 s of 460 nm CSTR irradiation
following the UV pulse) on the enzymatic systems AMC output (see
5.2.2 of the SI for details). [Cr] = 40 nM, [PI] = 200 μM, [AAPF-AMC]
= 80 μM. Flow rates: Cr, PI = 400 μL/h; AAPF-AMC, buffer = 200 μL/
h. Reactor volume = 196 μL. (B) Modeling of the behavior observed in
A, using a system of rate equations with experimentally determined rate
constants and roughly assumed photoswitching rates (see 7 of the SI).
(C) Impact of different UV pulse durations and pulse sequences (with
and without 10 s of 460 nm CSTR irradiation following a UV pulse) on
the Cr/Ap-based enzymatic systems AMC output (see 5.2.3 of the SI).
[Cr] = 20 nM, [PI] = 200 μM, [Z-Phe-Arg-AMC] = 100 μM, [Ap] =
0.1 U/mL. Flow rates: Cr, Ap = 200 μL/h; PI, Z-Phe-Arg-AMC = 400
μL/h; buffer = 100 μL/h. Reactor volume = 196 μL.
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selectively modulate the activity of enzymes within multi-
enzymatic systems.
output upon the next consecutive UV pulse. Thus, in the
absence of a blue light pulse, the temporal resolution of our
regulatory strategy is strictly limited by the underlying flow
properties. In contrast, a similar sequence with 5 s long blue
pulses after UV irradiation shows strong “filtering” of the UV
input pulses. The blue light immediately switches PI back to its
strongly inhibiting trans state, and infrequent pulses will thus not
lead to an appreciable buildup of active enzyme. Only when the
pulse offset is decreased to 2 s or shorter do we observe an
increase in AMC response. This is a first promising
demonstration that a combination of flow and reversible enzyme
activation yields a system with frequency filtering properties.
Stimulus Frequency Filtering in Out-of-Equilibrium
Enzymatic Systems by Incorporation of PIs. Finally, we
sought to characterize the temporal resolution of our strategy to
regulate the activity of enzymes under out-of-equilibrium
conditions (continuous addition and removal of PI, enzyme,
and buffer to/from CSTR) by applying different stimuli
frequencies to our system (Figure 5A). In two applied pulse
CONCLUSIONS
■
Ultrasensitivity is an emergent property of many biochemical
reaction networks. It enables these networks to generate switch-
like responses upon small changes in input parameters.
Introducing ultrasensitivity in synthetic systems is a desirable
goal in systems chemistry, as it would open up new routes to
control life-inspired, complex systems. However, ultrasensitivity
in biological systems is often the result of rather complex motifs,
which are challenging to recapitulate in synthetic systems. Here,
we have shown that enzymatic reactions operating under out-of-
equilibrium conditions generate an ultrasensitive response in a
surprisingly simple topology upon incorporation of photo-
switchable inhibitors. Introduction of a photoswitchable
inhibitor with sufficiently large differences in inhibitory
properties between the two photoisomers yielded switch-like
responses in enzymatic activity. Furthermore, we discovered
that by reversibly switching the photoisomers, even single
enzymatic reactions can filter out short pulses under flow
conditions. In the absence of photoinduced cis to trans
irradiation (blue light pulse), a period of over 30 min between
two UV light pulses was required to ensure an “on−off”
switching of the system, whereas short (5 s) blue light irradiation
immediately after UV pulses allowed us to detect pulses that
were only 5 s apart.
The simple building blocks introduced here set the scene for
more complex motifs that are now becoming feasible. We can
introduce photoswitchable inhibitors with different chromo-
phores, couple multiple enzymes into reaction networks, and
explore more complex motifs. The ability to modulate the
activity of individual enzymes in such networks using light will
provide an exceedingly versatile tool for creating ever more
advanced complex molecular systems.
Figure 5. (A) Impact of different UV pulse periodicity (pulse duration
= 30 s) and pulse sequences (with and without 5 s of 460 nm CSTR
irradiation following UV pulse) on the enzymatic systems AMC output
over time. [Cr] = 40 nM, [PI] = 200 μM, [AAPF-AMC] = 80 μM. Flow
rates: Cr, PI = 400 μL/h; AAPF-AMC, buffer = 200 μL/h. Reactor
volume = 196 μL. (B) Plot of the normalized change in AMC output of
the system as a function of the time offset between multiple UV pulses
(see 5.2.4 of the SI for details).
ASSOCIATED CONTENT
■
sequences, the offset between four UV pulses of 30 s duration
was gradually increased, ranging from as short as 1 s to 60 min. If
no blue light pulses are installed after UV irradiation, the PI
within the CSTR remains in its weaker inhibitory cis state due to
the very slow thermal cis to trans isomerization (t1/2 = 274 h).
Consecutively, the depicted exemplary [AMC]−t plot of the
pulse sequence without blue irradiation shows that the temporal
resolution of the system is insufficient to separate the four UV
pulses at higher pulse frequency, and they fuse into a single peak.
As a result, the maximum AMC concentration observed after
four UV pulses increases. Figure 5B shows the normalized
change in AMC output of the system as a function of the time
offset between multiple UV pulses (see 5.2.4 of the SI). In the
absence of blue light irradiation, a pulse offset of 30 min or
shorter is not sufficient to completely flush out all formed cis-PI
of the reactor, and the residual cis-PI leads to a higher AMC
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12956.
Experimental details, photophysical and enzymological
characterization data, copies of NMR spectra of new
compounds, simulation details, and assay procedures
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Wilhelm T. S. Huck − Institute for Molecules and Materials,
Radboud University Nijmegen, 6525 AJ Nijmegen, The
Netherlands; orcid.org/0000-0003-4222-5411;
Email: w.huck@science.ru.nl
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Journal of the American Chemical Society
Authors
Michael Teders − Institute for Molecules and Materials,
Radboud University Nijmegen, 6525 AJ Nijmegen, The
Netherlands
Aleksandr A. Pogodaev − Institute for Molecules and Materials,
Radboud University Nijmegen, 6525 AJ Nijmegen, The
Netherlands
Glenn Bojanov − Institute for Molecules and Materials,
Radboud University Nijmegen, 6525 AJ Nijmegen, The
Netherlands
pubs.acs.org/JACS
Article
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12956
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project has received funding from the European Union’s
Horizon 2020 research and innovation program under grant
agreement no. 862081 (CLASSY). Generous financial support
by the NWO (Spinoza prize to W.T.S.H.) and by the German
National Academy of Sciences Leopoldina (Postdoctoral
fellowship LPDS2020-04 to M.T.) is gratefully acknowledged.
We thank C. L. Fernández Regueiro, Dr. J. Taylor, Dr. O. R.
Maguire, and Dr. W. A. Velema (all Radboud University
Nijmegen) for fruitful discussions and experimental advice.
Furthermore, we would like to thank Prof. F. Glorius and F.
Sandfort (WWU Munster) for their support with mass
̈
spectrometric analysis of PIs.
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