Bioinspired Phosphatase-like Mimic Built from the Self-Assembly of de Novo Designed Helical Short Peptides

Article abstract of DOI:10.1021/acscatal.1c00129

Enzymes play vital roles in catalyzing biochemical reactions with high activity and selectivity, which is largely attributed to the delicately organized structure and groups at catalytic domains. Reconstruction of the enzymatic catalytic domains in artifi

Full text of DOI:10.1021/acscatal.1c00129

pubs.acs.org/acscatalysis
Research Article
Bioinspired Phosphatase-like Mimic Built from the Self-Assembly of
De Novo Designed Helical Short Peptides
Yutong Wang, Lijun Yang, Mengfan Wang,* Jiaxing Zhang, Wei Qi,* Rongxin Su, and Zhimin He
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ABSTRACT: Enzymes play vital roles in catalyzing biochemical
reactions with high activity and selectivity, which is largely attributed
to the delicately organized structure and groups at catalytic domains.
Reconstruction of the enzymatic catalytic domains in artificial systems, to
produce enzyme-like mimic, has become an attractive but challenging
subject. Herein, inspired by the helical structure in the catalytic center of
natural phosphatases, we created a phosphatase-like mimic through the
self-assembly of de novo designed helical heptapeptides. The helical
heptapeptides were elaborately decorated with well-studied catalytic
groups and exhibited obvious phosphatase-like catalytic activity upon
hierarchically self-assembling. In comparison with β-sheet-organized
peptide assemblies, we emphasized the significance of helical structure in
the hydrolysis of phosphoester bonds (over 1000 times higher in catalytic efficiency). The structure−activity relationship reveals that
the phosphatase-like function attributed to the specific helical dipole moment for the binding of substrates as well as the
supramolecular assembly for the formation of catalytic center. Moreover, we verified the feasibility of these helical species as a
potential substitute for adenosine triphosphatase (ATPase) and alkaline phosphatase (ALPase) in some specific biological processes.
This work not only presents an approach for the construction of artificial enzymes with simple peptide modules but also provides a
model for primitive enzymes formed from helical short peptides that relied on self-assembly to achieve a folded structure.
KEYWORDS: enzymatic mimics, helical peptide, phosphoester bond hydrolysis, de novo design, self-assembly
quite common in natural enzymes: domains with similar
peptide sequences, catalytic groups, or structures have the
same role in different systems.17 Fortunately, some principles
and methodologies have been developed to design peptides
with definite conformation or function.18−21 This inspired us
to create a simple peptide module, rather than an entire
protein or artificial material to replicate the structure of
catalytic domains and mimic the functions of natural enzymes.
Recently, the achievements of catalytic amyloids from β-
sheet peptide self-assembly show the way from concept to
practice. Through the rational design of assembling peptide
sequences, the catalytic domains of natural enzymes can be
replicated by simple peptide modules. For example,
Korendovych et al. designed a series of β-sheet-forming
heptapeptides and obtained a Zn(II)-coordinated structure
through the self-assembly of these peptides. The structure
replicated the catalytic center of natural carbonic anhydrase,
1. INTRODUCTION
As biocatalysts, enzymes have evolved into special substances
that play amazing roles in biological metabolism, nutrition, and
energy conversion, and they have shown outstanding
efficiency, specificity, and stereoselectivity in industrial fields.
As substitutes for natural enzymes, the construction of artificial
mimics with enzyme-like functions has become an exciting
field over the past decades, including synthetic peptides,1−3
nucleic acids,4−6 metal−organic complexes,7−9 and metal
oxides.10−12 Except for some ribonucleic acids, most enzymes
are proteins with well-organized three-dimensional structures.
Currently, based on the delicate structure of natural enzymes,
many supramolecular approaches have been developed for the
reconstruction of the enzymatic active sites in artificial
systems.13,14 Unfortunately, this approach is sometimes
arduous due to the insufficient understanding of the
structure−function relationship of enzymes and the chemical
differences between artificial materials and natural proteins.
More anticipated but even more challenging is the design of
a protein module with a predictable structure and catalytic
function.15,16 One of the philosophies from the engineering
field is the design and construction of standard parts or
components, which can perform specific tasks in different
contexts. At the protein level, this “plug-and-play” manner is
Received: January 11, 2021
Revised: April 9, 2021
Published: April 28, 2021
https://doi.org/10.1021/acscatal.1c00129
© 2021 American Chemical Society
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Scheme 1. (a) Catalytic Centers of Some Metal-Free Phosphoester Hydrolysis Enzymes (data from www.rcsb.org, PDB ID:
a
2I6P, 4GE2, 1K6M, and 1FPR); (b) Hierarchical Self-Assembly of Helical Peptides into Supramolecular Assemblies with
Phosphatase-like Activity
a
The N-terminal of the α-helix (green) points to the phosphate group (highlighted in blue) of the substrate (orange molecule).
showing catalytic ability toward the substrates of carbonic
anhydrase.22 Numata et al. constructed an enzyme-like catalyst
by introducing the classical serine-protease catalytic triad into
peptides that can self-assemble into amyloid-like fibrils. They
found that the formation of the β-sheet backbone is crucial in
organizing the protease-like active site.23
molecular assemblies (Scheme 1b). A hydrolytic reaction using
p-Nitrophenyl phosphate (pNPP) as a model substrate
confirmed the unique phosphatase-like catalytic activity of
helical assemblies. By comparing with β-sheet-organized
peptide assemblies, we highlighted the significance of helical
structure in phosphate ester hydrolysis. Based on the X-ray
diffraction analysis and computer simulation, the structure−
activity relationship was investigated to demonstrate the
function of the helical dipole moment and supramolecular
assembly in phosphatase-like catalysis. Moreover, we also
verified the feasibility of this helical species as a potential
substitute for adenosine triphosphatase (ATPase) and alkaline
phosphatase (ALPase) in specific biological processes. This
work presents a bioinspired phosphatase-like mimic via the
self-assembly of short helical peptides in the absence of any
metal cofactor and also shows the possibility of the
construction of artificial enzymes based on the self-assembly
of well-understood and completely de novo designed short
peptides.
Although many enzyme mimics have been realized based on
β-sheet peptides self-assembly, very little attention has been
paid to α-helix motifs. Actually, helical structure dominates
almost 30% of globular protein segments on average and takes
charge of many irreplaceable functions, such as DNA-binding
sites and proton channels.24,25 This prompted us to explore the
roles of helical structures in some natural enzymes and attempt
to design a similar helical module to execute supramolecular
assemblies with enzyme-like functions. It is well-known that
the spontaneous hydrolysis of phosphoester bonds is extremely
slow, with a cleavage half-life of ∼107 years.26 In living
organisms, the highly effective hydrolysis of phosphoester
bonds is related to various metal-containing or metal-free
phosphatase families,27 which play vital roles in energy
production, signal transduction, bone formation, and gene
maintenance. The protein crystal structure revealed that α-
helical motifs are ubiquitous in the catalytic domain of
phosphatase families,28 especially for those metal-free
phosphatases in which metal cofactors are absent in the active
site. The instinctive dipole of the helical structure promotes
the binding of negatively charged phosphate substrates to the
N-terminal of the helix and the consequent enhancing of the
attack toward phosphoester bonds (Scheme 1a).29 Inspired by
this, we wondered whether a self-assembled helical short
peptide could possess the phosphatase-mimicking active site
and exhibit phosphatase-like catalytic function.
2. RESULTS AND DISCUSSION
2.1. Peptide Sequence Design. To highlight the
significance of the helical structure for the phosphatase-
mimicking structure and function, a helical sequence that is as
simple as possible is required. Coiled coil is a structural motif
in proteins in which helixes are coiled together like the strands
of a rope. The peptide sequence of a coiled coil usually
followed the heptad repeat pattern of (hpphppp)n, or named
under abcdefg rule.31,32 In the repeat sequence, the a and d
positions are usually occupied by hydrophobic (h) residues,
and the b, c, e, f, and g positions are occupied by polar (p)
residues. In the folded state, the hydrophobic residues present
a periodic hydrophobic interface (a−d faces) and act as an
oligomerization domain during the association of two or more
such helical units. Driven by the a−d faces, the average 3.5-
residue spacing of hydrophobic side chains in heptad repeats is
very close to the typical 3.6-residue repeat of α-helix, resulting
in the formation of amphiphilic helices that are wrapped
around each other in a right-handed style (Figure 1). In a
recent study, Gazit et al. reported a heptapeptide (SHR-FF)
To achieve this goal, in this study, bioinspired de novo
design was performed to obtain a coiled-coil-forming helical
heptapeptide. As a unique association of helical peptides that
assemble into a superhelix structure, coiled coil often serves as
the basic module in many biological recognition processes.30
By installing catalytic residues into the helical heptapeptides,
we created a metal-free phosphatase mimic through the
hierarchical self-assembly of helical heptapeptides into supra-
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Based on the delicate electron-transfer system of the catalytic
triad in general hydrolases,35 the sequence involved the
decoration of serine (Ser, S) and histidine (His, H) residues
within the backbone as well as the carboxyl at the C-terminal.
Serine and histidine were assigned to the positions of b and f,
which hence has a minimal effect on helical folding. Taking the
orientation into consideration, two variants were assigned by
exchanging the positions of Ser and His at the b and f
positions, leading to the species of Hept-SH and Hept-HS
(Figure 1). Their influences on the structure and catalysis
ability of helical assemblies were also investigated in this study.
2.2. Peptide Assembly and Structural Investigation.
The designed peptides were obtained by standard solid-phase
synthesis, purified by reversed-phase high-performance liquid
chromatography (HPLC), and confirmed by electrospray
ionization (ESI) mass spectrometry (Figures S1 and S2). In
a typical procedure, the self-assembly of heptapeptides was
launched by dissolving a certain amount of heptapeptides into
dimethyl sulfoxide (DMSO), followed by mixing the peptide
stock with a neutral buffer. The fibrillar nanostructure in the
transmission electron microscopy (TEM) images implied the
well-organized self-assembly of Hept-SH and Hept-HS (Figure
2a,b). Circular dichroism (CD) was performed to investigate
the secondary structure of peptide molecules in the assemblies
(Figure 2c). The spectra displayed a double-negative Cotton
effect around 204 and 214 nm along with a positive peak at
196 nm, which is the characteristic pattern of the helical
conformation built by short peptides.36−38 Compared with the
spectrum of typical α-helix in proteins, the shorter length of
the helix results in a slight blue-shift of the characteristic peaks
by 3−7 nm and a lower intensity of the signals.39,40 This result
Figure 1. Design of Catalytic Helical Heptapeptides.
which is able to form coiled-coil assemblies.33 The peptide
sequence followed the typical abcdefg rule. In this study, a
similar helical heptapeptide was designed by integrating
hydrophobic phenylalanine (Phe, F) into the a and d positions
to form a “phenylalanine zipper”. To overcome the
thermodynamic barrier in helical folding, a noncoded amino
acid α-aminoisobutyric acid (Aib) was incorporated at the c, e,
and g positions. The additional α-methyl group in Aib limits
the ranges of allowed φ, ψ torsion angles compared with
alanine and imposes restrictions over the energetically
accessible conformational space, which thus stabilizes the
helical configuration.34 To guarantee the basic helical structure,
the heptapeptide sequence was minimally mutated with
catalytic residues for the purpose of enzyme-like catalysis.
Figure 2. TEM images of (a) Hept-SH and (b) Hept-HS assemblies. (c) CD spectra of the heptapeptides. (d) Different configurations of Hept-SH
and Hept-HS helices. (e) Torsion angles of the residues in Hept-SH (blue dots) and Hept-HS (yellow dots) superimposed over the ideal
Ramachandran plot.
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Figure 3. (a) Bond cleavage energies along with the hydrolysis reactions of pNPA and pNPP. Initial rate (V0) of the hydrolysis reaction as a
function of (b) pNPA or (c) pNPP concentration catalyzed by 0.08 mM helical assemblies. The data in (b) and (c) are smoothed fitting with the
Michaelis−Menten equation.
was also supported by attenuated total reflectance-infrared
Fourier transform infrared (ATR-FTIR) and Raman spectra
(Figures S7 and S8).41−43 The secondary structure assignment
based on amide I and amide III bands showed that helical
structures dominated the backbones of both heptapeptides.
NMR spectroscopy was employed to reveal the structures of
folded peptides in a solution.44,45 The strong and clear
nonsequential NOE constraints indicated the typical Hi−Hi+3
correlations within the heptapeptide molecules (Figures S9
and S10), which followed the character of 310-helix.46 X-ray
crystallography revealed the precise structures of the peptide
molecules. As shown in Figure 2d, the peptide backbone of
Hept-SH and Hept-HS displayed the typical helical config-
uration. Ramachandran plot indicated that the torsion angles
of all residues coincided well with the right-handed helical
configuration (Figure 2e and Tables S1 and S2).47 Unlike
SHR-FF, where water molecules bridge N−H and OC
groups via hydrogen bonds,33 Hept-SH and Hept-HS formed
three intramolecular hydrogen bonds directly between N−H
and OC groups located at the i and i+3 positions, which
promotes the stable folding of the helical structure (Figure
S11). When viewed from the N-terminal, the backbone of
heptapeptides exhibited a typical 310-helix configuration. Due
to the steric hindrance of imidazole in histidine, Hept-SH
formed a hydrogen bond between the g and c residues. For
Hept-HS, the histidine at the b position was replaced by serine,
leading to the formation of a hydrogen bond between a and d.
We speculated that this variation enhanced the hydrophobic or
π−π interactions between the phenylalanine side chains at the
a and d positions, which constrained Hept-HS to adopt a more
rigid helical folding than Hept-SH. Both heptapeptide helices
showed favorable thermal stability even though the temper-
ature rose to 90 °C (Figure S12).
2.3. Phosphatase-like Catalytic Ability. After demon-
strating the helical structure of the designed heptapeptides, we
further investigated the phosphatase-like catalytic ability of the
heptapeptide assemblies. For comparison, some previously
reported β-sheet-organized peptide assemblies were also
studied to investigate the different effects of the helical/β-
sheet structure on the hydrolysis of the phosphoester bond. It
is worth noting that although numerous β-sheet-organized
peptide assemblies claimed their hydrolase activity based on
the general hydrolytic substrate (p-nitrophenyl acetate, pNPA),
there is little evidence showing their catalytic ability toward
phosphoester substrates. Relative bond energy analysis
indicated that the cleavage of the phosphate ester bond in
pNPP requires more energy (109.96 kcal/mol) than destroying
the carboxylic ester bond in pNPA (97.88 kcal/mol) (Figure
3a). Thus, with respect to thermodynamics, the hydrolysis of
the phosphoester substrate is much more difficult than the
general substrate. Also, in organisms, the hydrolysis of these
two bonds adopts different pathways and enzyme families
(carboxylesterases versus phosphatases).48,49
Herein, the catalytic ability of helical assemblies toward both
the general hydrolytic substrate (pNPA) and the phosphate
ester substrate (pNPP) were evaluated. For the general
hydrolytic substrate pNPA, the initial hydrolysis rates V0-
pNPA as a function of various pNPA concentrations are shown
in Figure 3b. The reactions followed the typical Michaelis−
Menten model, which confirmed their enzyme-like catalytic
behavior. The catalytic rate constant of an enzyme (kcat) is the
maximal number of substrate molecules converted to product
per active site per unit time,50 which often reflects the speed
for the forward reaction of an enzyme−substrate complex.
Herein, kcat was obtained based on the linear Lineweaver−Burk
plots (Table 1 and Figure S13), indicating that Hept-SH
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nanofibers (kcat = 2397.39 23.08 × 10−5 s−1) are more active
than Hept-HS assemblies (kcat = 1888.79 23.08 × 10−5 s−1).
The hydrolytic ability of helical assemblies on pNPA comes
from the function of catalytic triad groups involved in the
peptide sequence. As a control, SHR-FF, which has been
reported to form suprahelical nanofibers in the study by Gazit,
displayed scarcely any catalytic activity, due to the lack of
catalytic residues.
For the phosphatase-like activity assay, we monitored the
product versus time plots for the hydrolysis of pNPP (Figure
S14). The result demonstrated that the addition of the Hept-
SH or Hept-HS assemblies significantly accelerated the
hydrolysis of pNPP. After 24 h, the pNP yielded the maximum
value and the turnover number (TON) reached 253 under the
catalysis of Hept-SH nanofibers. As the mutation of Hept-SH,
the helical assemblies are almost inactive toward pNPP once
the catalytic serine or histidine residue in Hept-SH sequence
was substituted by neutral residues, such as alanine (Figure
S15).
The hydrolysis of pNPP by Hept-SH or Hept-HS nanofibers
is also consistent with the enzymatic reaction kinetics (Figure
3c), and kcat of Hept-SH nanofibers (18.33 0.00 × 10−5 s−1)
is still higher than that of Hept-HS nanofibers (7.38 0.00 ×
10−5 s−1). Normally, the spontaneous hydrolysis of pNPP is
extremely slow (kuncat = 2.7 × 10−9 s−1).55 Here, the helical
assemblies showed a 104-fold acceleration than the uncatalyzed
reaction (Table S3), confirming the obvious phosphatase-
mimicking function of Hept-SH and Hept-HS nanofibers. We
also noticed that for both helical assemblies, the kcat values for
the pNPP hydrolysis are much lower than those for the pNPA
hydrolysis (Table 1). This result agrees with Arrhenius’ theory
(ln k = ln A − Ea/RT), where the reaction rate is inversely
proportional to the energy barrier.56 In addition, the Km values
for the hydrolysis of pNPP are much lower than those for the
hydrolysis of pNPA, which implied the superior affinity of
helical assemblies toward pNPP compared with pNPA. Besides
SHR-FF, we also detected the catalytic ability of Hept-AH and
Hept-SA assemblies in the hydrolysis of pNPP.
The comparison of catalytic performances of helical
assemblies with those of β-sheet and amyloid-like organizations
is more instructive. Catalytic efficiency (kcat/Km) is a
comprehensive parameter to evaluate the catalysis ability of
different enzymes toward a certain substrate. Herein, we
investigated the catalytic ability of SA-H, CoA-HSD, and Azo-
GFGH assemblies toward pNPP. These β-sheet peptide
assemblies have been proven to be efficient hydrolase mimics
in our previous reports (kcat/Km = 0.1−0.23 M−1 s−1)
according to the activity assay utilizing pNPA51,52 but
completely inactive toward pNPP (Figure S16). Similar
phenomenon occurred to the amyloid-like hydrolase mimic
F-Zn(II), which shows hyperactivity toward pNPA (kcat/Km =
76.54 M−1 s−1) but still inactive toward pNPP.53 The
significant reactivity of the helical species toward pNPP is
also recognized when compared with the previously reported
phosphatase mimic assembled from the β-sheet-organized
Lauryl-VVAGH-Am:54 the kcat/Km of helical assemblies for the
hydrolysis of pNPP are ∼1000 times higher than that of
Lauryl-VVAGH-Am assemblies (kcat/Km = 0.00069 M−1 s−1).
Therefore, we validated the phosphatase-like catalytic ability of
the helical assemblies and realized the superiority of the helical
structure over β-sheet in the hydrolysis of the phosphoester
bond.
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Figure 4. (a) Overall effect of the dipole moment (yellow arrow) in Hept-SH and Hept-HS helices obtained by the DFT calculation. The black
balls represent the α-carbon atoms in the peptide backbone. (b) Effect of the assembly time on the hydrolysis rate of pNPP catalyzed by helical
assemblies. Higher ordered organization patterns of (c) Hept-SH helices and (d) Hept-HS helices. The catalytic groups in (e) Hept-SH and (f)
Hept-HS assemblies. Dimers are differentiated by colors.
Admittedly, the phosphoester bond hydrolysis ability of the
helical assemblies are several orders of magnitude lower than
that of natural enzymes (e.g., the kcat of tyrosine phosphatase is
8.24 s−1 toward pNPP).57 However, it is still an exciting finding
because it demonstrated that the catalytic function of
phosphatase can be mimicked by simply replicating the helical
structure of the natural catalytic domain. The finding also gives
rise to the possibility that the earliest enzymes might have been
formed from short peptides or polymers with repeating amino
acid sequences that relied on self-assembly to achieve a folded
structure because the environment at the beginning of protein
evolution is too harsh for the biosynthesis of macromolecules
with delicate structure and cofactors.58,59 Eventually, with
respect to bioinspiration, the catalytic behaviors of the helical
short peptide assemblies confirmed the feasibility of
phosphatase-like supramolecular catalysis.
2.4. Structure−Activity Relationship. The different
catalytic performances of helical and β-sheet assemblies toward
phosphoester substrates reflected the different structure−
function relationships. In natural proteins, all of the hydrogen
bonds in α-helix motifs point to the same direction because the
peptide units align in the same orientation along the helical
axis. Peptide units have dipole moments that arise from the
different polarities of N−H and CO groups, and these
dipole moments are also aligned along the helical axis. The
overall effect is a significant net dipole for the α-helix, which
produces a partial positive charge at the amino end and a
partial negative charge at the carboxy end.60 For many
phosphatases, phosphate moieties are frequently found to
bind to the N-terminal of an α-helix (as illustrated in Scheme
1a), which corresponds to the optimal interaction between the
helix dipole and the phosphate group.28,61−63 Herein, the
density functional theory (DFT) calculation was carried out to
analyze the dipoles of the designed heptapeptide helices. As
shown in Figure 4a, although Hept-SH and Hept-HS folds are
not typical α-helix in configuration, dipoles were also found
along the helical axis in both helices. Molecular electrostatic
potential (MEP) analysis revealed that the N-terminal of the
helices is obviously positively charged (Figure S17), which
causes a strong electrostatic attraction toward the negatively
charged phosphate group. This interaction not only benefits
the orientation of the substrate but also helps to reduce the
potential energy of the phosphate group, making it prone to
attack from the nucleophile serine. The role of helical dipole
moment for phosphatase-like catalysis is also demonstrated by
the helical assemblies built from the N-terminal-acetyl or C-
terminal-amidated Hept-SH derivatives (Figure S18). In the β-
sheet structure, the dipole moments derived from intermo-
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Figure 5. (a) Two-step hydrolysis from ATP through ADP to AMP catalyzed by helical assemblies. (b) HPLC chromatography of the hydrolysis of
ATP. (c) Biomimetic hydroxyapatite mineralization reaction catalyzed by helical assemblies. (d−f) SEM images of mineral deposits on the surface
covered with Hept-SH assembly and (g−i) the blank surface. The scale bar is 1 μm. (j) EDX analysis of the mineralized nodules. (k) Quantification
of the deposited calcium on glass surfaces by the Alizarin Red S method.
lecular hydrogen bonds almost offset each other (Figure
S19).64 Unless specially designed, it is unusual for the β-sheet
structure to attract or reduce the potential energy of phosphate
substrates. We speculated that the electrostatic pattern of the
helix is highly conducive to Hept-SH species, in which the
serine is located exactly at the N-terminal of the helix, making
it more convenient to conduct a nucleophilic attack through its
deprotonated hydroxyl. However, for Hept-HS species, the
serine residue is located in the middle of the helix, which is a
less favorable configuration. This might explain the higher
catalytic efficiency of Hept-SH than that of Hept-HS.
The formation of supramolecular assemblies often contrib-
utes to the enzymatic enhancement for many catalytic
assemblies.65−67 In this study, we observed that the formation
of Hept-SH and Hept-HS assemblies is a time-dependent
process (Figures S20 and S21). Meanwhile, we detected the
catalytic activity during the assembly. Figure 4b shows that free
peptides are inactive on the first day (0 day), while the catalytic
ability (V0‑pNPP) starts to increase once primitive aggregates are
formed (1−2 day), and the activities reach the maximum when
the self-assembly is completed (after 5 days). A filtration
experiment was performed to separate the mature assemblies
from free monomers and oligomers, which also verified that
the assemblies contribute to the catalytic ability, rather than
the monomers or oligomers in the filtrate (Figure S22).
Besides, Figure S23 demonstrates that the free catalytic groups
(e.g., free imidazole, free His, Ser, Aib, or the combinations of
the amino acids) are inactive for the hydrolytic reaction, which
verified that the simple mixture of active residues is invalid for
catalysis. In addition, the sedimentation experiment further
demonstrates that the well-ordered self-assembling is essential
for the active species because the amorphous peptide floccules
are inactive whether they are incubated or not (Figure S24).
These results reflect that the phosphatase-like activity is closely
associated with the formation of well-ordered supramolecular
assemblies.
Single-crystal X-ray diffraction analysis demonstrated the
hierarchical organization of heptapeptide helices within the
assemblies. For both heptapeptides, the twin helical monomers
interacted with each other to form coiled-coil dimers in a
parallel orientation, driven by the hydrophobic and aromatic
interactions of the phenylalanine zipper within the a−d face. It
is observed that the Hept-SH helices interlace tighter in the
dimer than Hept-HS because of the stretchable phenylalanine
side chains within the a−d face. In contrast, the phenylalanine
side chains in Hept-HS blocked the close packing of twin
helices due to the restriction of intramolecular hydrogen bonds
(Figure S25).
Due to the different arrangement of the helical dimer, the
dimeric units adopt a different packing pattern when they
propagate to form a continuous array of supramolecular
assemblies. For Hept-SH, the dimeric unit repeats itself along
the a-axis and b-axis (Figure S26, note: the angle between the
a-axis and b-axis is 120°). While in the c-direction, the Hept-
SH dimer rotates twice around the c-axis, forming a 3-dimer
stack with an angle of 120° (Figure 4c). As a result, the dimers
formed a cylinder along the c-axis, and these cylinders further
organized into high-ordered assemblies. Unlike Hept-SH
dimers, the twin Hept-HS helices formed “/ \”-type dimers,
which leads to the alternate packing of antiparallel dimers
along the c-axis (Figure 4d). This arrangement was repeated
and well-aligned in direction a but staggered from the view of
the b-direction (Figure S27), which exhibited a completely
different packing mode from Hept-SH assemblies. For both
assemblies, the well-ordered aggregations of dimers are
stabilized either through the hydrogen bonds between the
head-to-tail backbone or the hydrogen bonds within the
hydrophilic faces. Additionally, upon self-assembly, the
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histidine residue, serine residue, and carboxyl at the C-terminal
from different dimers approach each other spatially, which is
favorable to form a triad for substrate binding and catalysis
(Figure 4e,f).
ing the production of HAP (Figure 5j). As a control, only a
small amount of irregular precipitates was formed on the blank
glass surface (Figure 5g−i). The Alizarin Red S method was
performed to quantify the amount of calcium deposited on
glass surfaces. As shown in Figure 5k, the deposited calcium
level on the Hept-SH-coated surface increased much faster
than that on the blank surface, confirming the ALPase-like
catalytic ability of Hept-SH species, which accelerated the
hydrolysis of β-glycerophosphate.
Based on the crystal structure of supramolecular assemblies,
molecule docking was carried out to get an insight into the
binding of pNPP in the catalytic domain (Figure S28). For
Hept-SH assemblies, the phosphate group embedded into the
gap between helical dimers, binding to His4 and the C-
terminal carboxyl (Aib7) through intermolecular hydrogen
bonds within 4 Å, which implied the possible catalytic
mechanism through the electrical transfer among the serine
residue, histidine residue and carboxyl at the C-terminal.
Remarkably, it was found that the substrate-bonded residues
came from different helical dimers, which indicated the
importance of supramolecular self-assembly in the formation
of the catalytic center. Due to the combination of the helical
structure and catalytic triad, the Hept-SH assemblies displayed
favorable hydrolytic ability toward pNPP. For Hept-HS
assemblies, pNPP suffered from steric hindrance within the
array of antiparallel helical dimers. The distance between the
phosphate group and the catalytic residues was farther (6−10
Å), leading to the inferior catalytic performance of Hept-HS
assemblies compared with that of Hept-SH assemblies.
2.5. Catalytic Function as ATPase and ALPase Mimics.
Using pNPP as the model substrate, we have verified the
competence of helical assemblies in the hydrolysis of the
phosphoester bond in the absence of any metal cofactor. Based
on the common existence of helical domains in the active
center of phosphatase family, we further investigated the
potential of this de novo designed catalytic module as specific
phosphatase mimics in physiological reactions.
ATPase is a class of enzymes that catalyze the two-step
hydrolysis of adenosine triphosphate (ATP) into adenosine
diphosphate (ADP) and adenosine monophosphate (AMP).
The breaking of phosphoester bonds produces energy that is
required by many physiological processes (Figure 5a). The
HPLC result indicated that Hept-SH and Hept-HS assemblies
exhibited an obvious ATPase-like activity in the conversion of
ATP → ADP → AMP (Figure 5b). Besides, Hept-SH
assemblies exhibited superior catalytic activity compared with
Hept-HS assemblies. A total of 69.13% of ATP was hydrolyzed
by Hept-SH after 36 h, whereas only 44.05% hydrolyzation was
achieved by Hept-HS. This result is consistent with the activity
evaluation using pNPP as a substrate.
Bone formation is a complex process that requires ALPase at
each level of this process. ALPase increases the inorganic
phosphate/pyrophosphate ratio in the extracellular matrix
through the nonspecific cleavage of phosphate esters. This
reaction elevates the local concentration of inorganic
phosphate and promotes the formation of hydroxyapatite
(HAP, Ca5(PO4)3(OH)), which is an essential building block
of bone and teeth (Figure 5c). Considering the phosphatase-
like catalytic ability of Hept-SH assemblies, we envisage that
they have great potential to simulate the osteogenic function of
natural ALPase. To verify this, glass slides were coated with
Hept-SH assemblies and then incubated in a solution
containing β-glycerophosphate and CaCl2. SEM analysis
(Figure 5d−f) revealed that during the incubation, nodal-like
mineral deposits were gradually generated on the surface
covered by Hept-SH. Energy-dispersive X-ray (EDX) spectral
analysis indicated high calcium and phosphorus levels of
mineralized nodules on the Hept-SH-coated surface, confirm-
3. CONCLUSIONS
After millions of years of evolution, existing enzymes are
composed of at least hundreds of amino acid residues.
Polypeptides with long sequences are thought to be able to
form complicated structures upon molecular folding and thus
possess recognition, accumulation, and catalytic functions.
However, this consensus may be challenged by catalytic
peptide assemblies. Many β-sheet-forming peptides, from
tripeptides to 20-residue peptides, have been reported to be
capable of catalyzing simple reactions under physiological
conditions, through self-assembly with or without cofactors.
These findings give rise to the possibility that short peptides
with amyloid structures were the “primitive enzymes” on the
early earth. In the case of helix-forming peptides, although
helical peptides or coiled-coil bundles have also been used in
the organization of enzyme-like catalysts, they merely act as the
scaffolds for supporting catalytic residues or metal cofac-
tors,68,69 whereas the intrinsic features of the helical structure
in enzymatic reactions are almost ignored. In this study,
inspired by the helical structure in the metal-free phosphatase
family, we have shown the de novo design and construction of
a phosphatase mimic through the self-assembly of helical
heptapeptides. In comparison with the β-sheet-organized
species, we demonstrated the unique helical assemblies in
the hydrolysis of the phosphoester bonds due to the helical-
derived dipole moment and the supramolecular assembly. This
study not only presented a strategy for the construction of
phosphatase-like mimics but also extended the understanding
of the role of helical structure in the evolution of primitive
enzymes.
4. MATERIALS AND METHODS
4.1. Materials. Lyophilized heptapeptides (Hept-SH and
Hept-HS) were purchased from the Top-Peptide Biotechnol-
ogy (Shanghai) Co. Ltd. p-Nitrophenyl phosphate (pNPP), p-
nitrophenyl acetate (pNPA), 5′-adenosine triphosphate
disodium (ATP), β-glycerophosphate (BGP), and Alizarin
Red S were purchased from the Aladdin Industrial Corp.
(Shanghai, China). Other chemicals and solvents of analytical
grade were obtained from commercial sources.
4.2. Preparation of Heptapeptide Self-Assemblies.
Fresh peptide stock solutions were prepared by dissolving 3
μmol of heptapeptide in 20 μL of DMSO and diluting the
solution with 980 μL of Tris−HCl buffer (10 mM, pH7.4) to a
final peptide concentration of 3 mM. Then, the solutions were
thoroughly mixed and incubated at room temperature.
4.3. Catalytic Reaction and Kinetic Assays. In a typical
experiment of pNPP hydrolysis, 3 mM incubated peptide stock
solution was diluted with Tris−HCl buffer (25 mM, pH7.4),
and freshly prepared pNPP stock solution (dissolved in
ddH2O) under different concentrations was added before the
measurement of absorbance at 400 nm. The final concen-
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pubs.acs.org/acscatalysis
Research Article
tration of the peptide is 0.08 mM, and the concentration of
pNPP is 0.1−8 mM. Two hundred microliters of the reactant
was added in a 96-well microplate well for a time-dependent
absorbance reading under 37 °C. The data was background
corrected (self-hydrolyzation without peptide catalysts), and
errors were obtained from the standard deviation of three
repeats. The hydrolysis of pNPA was carried out under the
same peptide/substrate concentration, and the pNPA stock
solution was dissolved in acetonitrile.
Enzymatic kinetic assays were studied by fitting the
Michaelis−Menten equation to give a three-state curve as
well as kinetic parameters.70 To obtain accurate Km and kcat
values, Lineweaver−Burk plots were performed to calculated
kinetic parameters (peptide assemblies 0.08 mM, substrate
0.9−8 mM).71
4.4. Transmission Electron Microscopy (TEM). Mor-
phologies of peptide self-assemblies were collected using a
JEM-2100F (JEOL Ltd., Japan) electron microscope under
200 kV. Five microliters of an aliquot of the stock solution of
peptide assembly was used for the sample preparation on 200
meshes carbon-coated copper grid, and the sample was
negatively stained with 2% uranyl acetate.
the osteogenic solution was removed from the surface and
replaced with a fresh one. After 6 days, the supernatant was
removed, and the slides were rinsed with ddH2O 3 times and
air-dried. The slides were stained with 0.1% Alizarin Red S for
20 min to quantify the mineralized calcium. After that, the
slides were washed with ddH2O 3 times, and the calcium-
bound dye was then extracted by 10% cetylpyridinium chloride
for 20 min. Finally, the mineralized calcium can be estimated
by directly determining the absorbance of Alizarin Red S at
562 nm using a UV−vis spectrophotometer (TU-1900, Persee
Devices, China).
The mineralized slides are directly dried in air and sputter-
coated with platinum before scanning electron microscopy
(SEM) characterization. Images were recorded on an S-4800
field emission SEM (Hitachi High-Technologies Co., Japan) at
an acceleration voltage of 3 kV, and the element analysis was
conducted on the EDX module.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.1c00129.
4.5. Circular Dichroism (CD). In CD experiments, DMSO
used in the peptide assembly preparation was substituted by
HFIP to eliminate the background signal. The assembled
peptides were diluted to 0.75 mM and tested in a 0.1 mm path
length quartz cuvette. Circular dichroism spectra in the range
of 190−260 nm were recorded on a J-810 CD spectropo-
larimeter (JASCO, Japan). The background-subtracted data
was converted from ellipticity (deg) to molar ellipticity (MER;
deg cm2 dmol−1 res−1) by normalizing for the peptide bond
concentration and the cell path length. The temperature
stability of the peptide secondary structure was tested between
20 and 90 °C with a heating−cooling program. After reaching
the desired temperature, the samples were incubated for 10
min before spectral scanning.
4.6. Crystal Structure Analysis. Colorless diffraction
quality crystals were grown by slowly evaporating solvent from
solutions containing 3 mM peptide at room temperature.
Single-crystal X-ray diffraction data were collected on a Bruker
SMART APEX II diffractometer with Cu Kα1 (λ = 1.5418 Å)
radiation. The structure was refined by full-matrix least-squares
against F2 with SHELXL-2013. The crystallographic data are
summarized in Table S4. The CIF for each structure is
uploaded in the Supporting Information.
4.7. ATP Hydrolysis. Assembled heptapeptide stocks were
dispersed in Tris−HCl buffer (10 mM, pH 7.4) to a final
concentration of 0.5 mM, and the reaction concentration of
the substrate 5′-adenosine triphosphate disodium (ATP) was 1
mM. The reaction was kept at 25 °C. During the reaction, the
composition of the reactant was measured using high-
performance liquid chromatography (LC-20A, Shimadzu,
Japan) equipped with a reverse-phase C18 column. The eluent
was a potassium phosphate buffer (50 mM, pH 6.5) with a
flow rate of 0.8 mL min−1, and the detection wavelength was
254 nm using a UV detector.
4.8. Biomineralization. Clean glass slides were coated
with 0.5 mM Hept-SH peptide stock solution, and the
mineralization was conducted in 24-well plates. In each well,
800 μL of osteogenic solution containing 15 mM of β-
glycerophosphate (Pi source) and 20 mM of CaCl2 (Ca2+
source) in Tris−HCl buffer (25 mM, pH 7.4) was added.
Incubation was conducted at room temperature. Once a day,
Additional methods of secondary structure character-
ization, computational calculation, AFM, pyrene fluo-
rescence, and sedimentation experiment; Supporting
data, figures, and tables about molecular characterization
(HPLC and ESI-MS), secondary structure (FTIR,
Raman, NMR, and CD), kinetic studies, active studies,
calculation results, and crystal information (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Mengfan Wang − School of Chemical Engineering and
Technology, State Key Laboratory of Chemical Engineering,
Tianjin University, Tianjin 300350, P. R. China; Tianjin
Key Laboratory of Membrane Science and Desalination
Technology, Tianjin 300350, P. R. China; Email: mwang@
tju.edu.cn
Wei Qi − School of Chemical Engineering and Technology,
State Key Laboratory of Chemical Engineering, Tianjin
University, Tianjin 300350, P. R. China; The Co-Innovation
Centre of Chemistry and Chemical Engineering of Tianjin,
Tianjin 300072, P. R. China; Tianjin Key Laboratory of
Membrane Science and Desalination Technology, Tianjin
300350, P. R. China; orcid.org/0000-0002-7378-1392;
Email: qiwei@tju.edu.cn
Authors
Yutong Wang − School of Chemical Engineering and
Technology, State Key Laboratory of Chemical Engineering,
Tianjin University, Tianjin 300350, P. R. China
Lijun Yang − School of Chemical Engineering and Technology,
State Key Laboratory of Chemical Engineering, Tianjin
University, Tianjin 300350, P. R. China
Jiaxing Zhang − School of Chemical Engineering and
Technology, State Key Laboratory of Chemical Engineering,
Tianjin University, Tianjin 300350, P. R. China
Rongxin Su − School of Chemical Engineering and
Technology, State Key Laboratory of Chemical Engineering,
Tianjin University, Tianjin 300350, P. R. China; The Co-
Innovation Centre of Chemistry and Chemical Engineering of
Tianjin, Tianjin 300072, P. R. China; Tianjin Key
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Research Article
(13) Yao, X.; Liu, T.; Xie, Y.; Chu, Z.; Jin, W. In Situ-Forming
Magnetic Fe3O4 Nanoroses on Defect-Controllable Mesoporous
Graphene Oxide for Enzyme-Mimic Sensing. Ind. Eng. Chem. Res.
2020, 59, 17934−17943.
(14) He, Q.; Kelliher, M.; Bähring, S.; Lynch, V. M.; Sessler, J. L. A
Bis-calix[4]pyrrole Enzyme Mimic That Constrains Two Oxoanions
in Close Proximity. J. Am. Chem. Soc. 2017, 139, 7140−7143.
(15) Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.;
Woolfson, D. N. Installing hydrolytic activity into a completely de
novo protein framework. Nat. Chem. 2016, 8, 837−844.
(16) Liu, Q.; Wan, K.; Shang, Y.; Wang, Z.-G.; Zhang, Y.; Dai, L.;
Wang, C.; Wang, H.; Shi, X.; Liu, D.; Ding, B. Cofactor-free oxidase-
mimetic nanomaterials from self-assembled histidine-rich peptides.
Nat. Mater. 2021, 20, 395−402.
(17) Nothling, M. D.; Xiao, Z.; Bhaskaran, A.; Blyth, M. T.; Bennett,
C. W.; Coote, M. L.; Connal, L. A. Synthetic Catalysts Inspired by
Hydrolytic Enzymes. ACS Catal. 2019, 9, 168−187.
(18) Metrano, A. J.; Abascal, N. C.; Mercado, B. Q.; Paulson, E. K.;
Hurtley, A. E.; Miller, S. J. Diversity of Secondary Structure in
Catalytic Peptides with β-Turn-Biased Sequences. J. Am. Chem. Soc.
2017, 139, 492−516.
(19) Colombo, G.; Soto, P.; Gazit, E. Peptide self-assembly at the
nanoscale: a challenging target for computational and experimental
biotechnology. Trends Biotechnol. 2007, 25, 211−218.
(20) Wang, J.; Liu, K.; Xing, R.; Yan, X. Peptide self-assembly:
thermodynamics and kinetics. Chem. Soc. Rev. 2016, 45, 5589−5604.
(21) Rhys, G. G.; Wood, C. W.; Beesley, J. L.; Zaccai, N. R.; Burton,
A. J.; Brady, R. L.; Thomson, A. R.; Woolfson, D. N. Navigating the
Structural Landscape of De Novo α-Helical Bundles. J. Am. Chem. Soc.
2019, 141, 8787−8797.
(22) Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stöhr, J.; Smith, T. A.;
Hu, X.; DeGrado, W. F.; Korendovych, I. V. Short peptides self-
assemble to produce catalytic amyloids. Nat. Chem. 2014, 6, 303.
(23) Wong, Y.-M.; Masunaga, H.; Chuah, J.-A.; Sudesh, K.; Numata,
K. Enzyme-Mimic Peptide Assembly To Achieve Amidolytic Activity.
Biomacromolecules 2016, 17, 3375−3385.
(24) Mou, Y.; Yu, J.-Y.; Wannier, T. M.; Guo, C.-L.; Mayo, S. L.
Computational design of co-assembling protein−DNA nanowires.
Nature 2015, 525, 230−233.
Laboratory of Membrane Science and Desalination
Technology, Tianjin 300350, P. R. China; orcid.org/
0000-0001-9778-9113
Zhimin He − School of Chemical Engineering and Technology,
State Key Laboratory of Chemical Engineering, Tianjin
University, Tianjin 300350, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.1c00129
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (21621004 and 21676191). The
authors gratefully acknowledge Dr. Lin Zhi from the School of
Life Sciences, Tianjin University, for his valuable advices on
the NMR analysis.
REFERENCES
■
(1) Lewis, C. A.; Gustafson, J. L.; Chiu, A.; Balsells, J.; Pollard, D.;
Murry, J.; Reamer, R. A.; Hansen, K. B.; Miller, S. J. A Case of Remote
Asymmetric Induction in the Peptide-Catalyzed Desymmetrization of
a Bis(phenol). J. Am. Chem. Soc. 2008, 130, 16358−16365.
(2) Metrano, A. J.; Chinn, A. J.; Shugrue, C. R.; Stone, E. A.; Kim,
B.; Miller, S. J. Asymmetric Catalysis Mediated by Synthetic Peptides,
Version 2.0: Expansion of Scope and Mechanisms. Chem. Rev. 2020,
120, 11479−11615.
(3) Zozulia, O.; Dolan, M. A.; Korendovych, I. V. Catalytic peptide
assemblies. Chem. Soc. Rev. 2018, 47, 3621−3639.
(4) Biniuri, Y.; Albada, B.; Wolff, M.; Golub, E.; Gelman, D.;
Willner, I. Cu2+ or Fe3+ Terpyridine/Aptamer Conjugates: Nucle-
oapzymes Catalyzing the Oxidation of Dopamine to Aminochrome.
ACS Catal. 2018, 8, 1802−1809.
(5) Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem.
Rev. 2009, 109, 1948−1998.
(6) Silverman, S. K. Catalytic DNA: Scope, Applications, and
Biochemistry of Deoxyribozymes. Trends Biochem. Sci. 2016, 41, 595−
609.
(25) Li, F.; Li, Y.; Zhou, Z.; Lv, S.; Deng, Q.; Xu, X.; Yin, L.
Engineering the aromaticity of cationic helical polypeptides toward
“self-activated” DNA/siRNA delivery. ACS Appl. Mater. Interfaces
2017, 9, 23586−23601.
(7) Xu, B.; Wang, H.; Wang, W.; Gao, L.; Li, S.; Pan, X.; Wang, H.;
Yang, H.; Meng, X.; Wu, Q.; Zheng, L.; Chen, S.; Shi, X.; Fan, K.;
Yan, X.; Liu, H. A Single-Atom Nanozyme for Wound Disinfection
Applications. Angew. Chem., Int. Ed. 2019, 58, 4911−4916.
(8) Luo, L.; Huang, L.; Liu, X.; Zhang, W.; Yao, X.; Dou, L.; Zhang,
X.; Nian, Y.; Sun, J.; Wang, J. Mixed-Valence Ce-BPyDC Metal−
Organic Framework with Dual Enzyme-like Activities for Colori-
metric Biosensing. Inorg. Chem. 2019, 58, 11382−11388.
(9) Wang, D.; Wu, H.; Lim, W. Q.; Phua, S. Z. F.; Xu, P.; Chen, Q.;
Guo, Z.; Zhao, Y. A Mesoporous Nanoenzyme Derived from Metal−
Organic Frameworks with Endogenous Oxygen Generation to
Alleviate Tumor Hypoxia for Significantly Enhanced Photodynamic
Therapy. Adv. Mater. 2019, 31, No. 1901893.
(10) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.;
Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Intrinsic peroxidase-
like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2,
577−583.
(11) Natalio, F.; André, R.; Hartog, A. F.; Stoll, B.; Jochum, K. P.;
Wever, R.; Tremel, W. Vanadium pentoxide nanoparticles mimic
vanadium haloperoxidases and thwart biofilm formation. Nat.
Nanotechnol. 2012, 7, 530−535.
(26) Wolfenden, R. Benchmark reaction rates, the stability of
biological molecules in water, and the evolution of catalytic power in
enzymes. Annu. Rev. Biochem. 2011, 80, 645−667.
(27) Yu, Z.-H.; Zhang, Z.-Y. Regulatory Mechanisms and Novel
Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases.
Chem. Rev. 2018, 118, 1069−1091.
(28) Hirsch, A. K.; Fischer, F. R.; Diederich, F. Phosphate
recognition in structural biology. Angew. Chem., Int. Ed. 2007, 46,
338−352.
(29) Dillet, V.; Van Etten, R. L.; Bashford, D. Stabilization of
Charges and Protonation States in the Active Site of the Protein
Tyrosine Phosphatases: A Computational Study. J. Phys. Chem. B
2000, 104, 11321−11333.
̌ ̌ ̌
(30) Ljubetic, A.; Lapenta, F.; Gradisar, H.; Drobnak, I.; Aupic, J.;
̌
̌
̌
̌
̌
Strmsek, Z.; Lainscek, D.; Hafner-Bratkovic, I.; Majerle, A.; Krivec,
́
̌
̌
́
N.; Bencina, M.; Pisanski, T.; Velickovic, T. C.; Round, A.; Carazo, J.
M.; Melero, R.; Jerala, R. Design of coiled-coil protein-origami cages
that self-assemble in vitro and in vivo. Nat. Biotechnol. 2017, 35,
1094−1101.
(31) Lupas, A. Coiled coils: new structures and new functions.
Trends Biochem. Sci. 1996, 21, 375−382.
(12) Singh, N.; Savanur, M. A.; Srivastava, S.; D’Silva, P.; Mugesh,
G. A Redox Modulatory Mn3O4 Nanozyme with Multi-Enzyme
Activity Provides Efficient Cytoprotection to Human Cells in a
Parkinson’s Disease Model. Angew. Chem., Int. Ed. 2017, 56, 14267−
14271.
(32) Woolfson, D. N. In Fibrous Proteins: Structures and Mechanisms;
Parry, D. A. D.; Squire, J. M., Eds.; Springer International Publishing:
Cham, 2017; pp 35−61.
(33) Mondal, S.; Adler-Abramovich, L.; Lampel, A.; Bram, Y.;
Lipstman, S.; Gazit, E. Formation of functional super-helical
5848
https://doi.org/10.1021/acscatal.1c00129
ACS Catal. 2021, 11, 5839−5849

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
assemblies by constrained single heptad repeat. Nat. Commun. 2015,
6, No. 8615.
(52) Zhao, Y.; Lei, B.; Wang, M.; Wu, S.; Qi, W.; Su, R.; He, Z. A
supramolecular approach to construct a hydrolase mimic with photo-
switchable catalytic activity. J. Mater. Chem. B 2018, 6, 2444−2449.
(53) Makam, P.; Yamijala, S. S.; Tao, K.; Shimon, L. J.; Eisenberg, D.
S.; Sawaya, M. R.; Wong, B. M.; Gazit, E. Non-proteinaceous
hydrolase comprised of a phenylalanine metallo-supramolecular
amyloid-like structure. Nat. Catal. 2019, 2, 977−985.
(54) Gulseren, G.; Yasa, I. C.; Ustahuseyin, O.; Tekin, E. D.;
Tekinay, A. B.; Guler, M. O. Alkaline phosphatase-mimicking peptide
nanofibers for osteogenic differentiation. Biomacromolecules 2015, 16,
2198−2208.
(55) Olguin, L. F.; Askew, S. E.; O’Donoghue, A. C.; Hollfelder, F.
Efficient Catalytic Promiscuity in an Enzyme Superfamily: An
Arylsulfatase Shows a Rate Acceleration of 1013 for Phosphate
Monoester Hydrolysis. J. Am. Chem. Soc. 2008, 130, 16547−16555.
(56) Menzinger, M.; Wolfgang, R. The meaning and use of the
Arrhenius activation energy. Angew. Chem., Int. Ed. 1969, 8, 438−444.
(57) Madan, L. L.; Gopal, B. Conformational Basis for Substrate
Recruitment in Protein Tyrosine Phosphatase 10D. Biochemistry
2011, 50, 10114−10125.
(58) Romero Romero, M. L.; Rabin, A.; Tawfik, D. S. Functional
Proteins from Short Peptides: Dayhoff’s Hypothesis Turns 50. Angew.
Chem., Int. Ed. 2016, 55, 15966−15971.
(59) Greenwald, J.; Kwiatkowski, W.; Riek, R. Peptide Amyloids in
the Origin of Life. J. Mol. Biol. 2018, 430, 3735−3750.
(60) Ganesan, S. J.; Matysiak, S. Role of Backbone Dipole
Interactions in the Formation of Secondary and Supersecondary
Structures of Proteins. J. Chem. Theory. Comput. 2014, 10, 2569−
2576.
(34) Jones, J. E.; Diemer, V.; Adam, C.; Raftery, J.; Ruscoe, R. E.;
Sengel, J. T.; Wallace, M. I.; Bader, A.; Cockroft, S. L.; Clayden, J.;
Webb, S. J. Length-Dependent Formation of Transmembrane Pores
by 310-Helical α-Aminoisobutyric Acid Foldamers. J. Am. Chem. Soc.
2016, 138, 688−695.
(35) Buller, A. R.; Townsend, C. A. Intrinsic evolutionary constraints
on protease structure, enzyme acylation, and the identity of the
catalytic triad. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E653.
(36) Miles, A. J.; Wallace, B. A. Circular dichroism spectroscopy of
membrane proteins. Chem. Soc. Rev. 2016, 45, 4859−4872.
(37) Mondal, S.; Adler-Abramovich, L.; Lampel, A.; Bram, Y.;
Lipstman, S.; Gazit, E. Formation of functional super-helical
assemblies by constrained single heptad repeat. Nat. Commun.
2015, 6, No. 8615.
(38) Goodman, M.; Toniolo, C.; Peggion, E. Conformational
aspects of polypeptides. XXIX. Conformationl assignments for some
aromatic polypeptides by far-uv cotton effects. New results.
Biopolymers 1968, 6, 1691−1695.
(39) Chin, D.-H.; Woody, R. W.; Rohl, C. A.; Baldwin, R. L. Circular
dichroism spectra of short, fixed-nucleus alanine helices. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 15416.
(40) Shepherd, N. E.; Hoang, H. N.; Abbenante, G.; Fairlie, D. P.
Single Turn Peptide Alpha Helices with Exceptional Stability in
Water. J. Am. Chem. Soc. 2005, 127, 2974−2983.
(41) Chen, Y.; Viereck, J.; Harmer, R.; Rangan, S.; Bartynski, R. A.;
Galoppini, E. Helical Peptides Design for Molecular Dipoles
Functionalization of Wide Band Gap Oxides. J. Am. Chem. Soc.
2020, 142, 3489−3498.
(42) Ji, W.; Yuan, C.; Chakraborty, P.; Gilead, S.; Yan, X.; Gazit, E.
Stoichiometry-controlled secondary structure transition of amyloid-
derived supramolecular dipeptide co-assemblies. Commun. Chem.
2019, 2, No. 65.
(43) Dolui, S.; Mondal, A.; Roy, A.; Pal, U.; Das, S.; Saha, A.; Maiti,
N. C. Order, Disorder, and Reorder State of Lysozyme: Aggregation
Mechanism by Raman Spectroscopy. J. Phys. Chem. B 2020, 124, 50−
60.
(44) Polizzi, N. F.; Wu, Y.; Lemmin, T.; Maxwell, A. M.; Zhang, S.-
Q.; Rawson, J.; Beratan, D. N.; Therien, M. J.; DeGrado, W. F. De
novo design of a hyperstable non-natural protein−ligand complex
with sub-Å accuracy. Nat. Chem. 2017, 9, 1157−1164.
(45) Ulas, G.; Lemmin, T.; Wu, Y.; Gassner, G. T.; DeGrado, W. F.
Designed metalloprotein stabilizes a semiquinone radical. Nat. Chem.
2016, 8, 354−359.
(46) Gratias, R.; Konat, R.; Kessler, H.; Crisma, M.; Valle, G.;
Polese, A.; Formaggio, F.; Toniolo, C.; Broxterman, Q. B.; Kamphuis,
J. First Step Toward the Quantitative Identification of Peptide 310-
Helix Conformation with NMR Spectroscopy: NMR and X-ray
Diffraction Structural Analysis of a Fully-Developed 310-Helical
Peptide Standard. J. Am. Chem. Soc. 1998, 120, 4763−4770.
(47) Mensch, C.; Barron, L. D.; Johannessen, C. Ramachandran
mapping of peptide conformation using a large database of computed
Raman and Raman optical activity spectra. Phys. Chem. Chem. Phys.
2016, 18, 31757−31768.
(48) Ganguly, A.; Luong, T. Q.; Brylski, O.; Dirkmann, M.; Möller,
D.; Ebbinghaus, S.; Schulz, F.; Winter, R.; Sanchez-Garcia, E.; Thiel,
W. Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger
Hydrolase. J. Phys. Chem. B 2017, 121, 6390−6398.
(49) Cleland, W. W.; Hengge, A. C. Enzymatic mechanisms of
phosphate and sulfate transfer. Chem. Rev. 2006, 106, 3252−3278.
(50) Roskoski, R. Michaelis-Menten Kinetics. In Reference Module in
Biomedical Sciences 2015.
́
(61) Longo, L. M.; Petrovic, D.; Kamerlin, S. C. L.; Tawfik, D. S.
Short and simple sequences favored the emergence of N-helix
phospho-ligand binding sites in the first enzymes. Proc. Natl. Acad. Sci.
U.S.A. 2020, 117, 5310.
́
(62) Petrovic, D.; Risso, V. A.; Kamerlin, S. C. L.; Sanchez-Ruiz, J.
M. Conformational dynamics and enzyme evolution. J. R. Soc.,
Interface 2018, 15, No. 20180330.
(63) Querol-García, J.; Fernández, F. J.; Marin, A. V.; Gómez, S.;
̀
Fulla, D.; Melchor-Tafur, C.; Franco-Hidalgo, V.; Albertí, S.;
Juanhuix, J.; Rodríguez de Córdoba, S.; Regueiro, J. R.; Vega, M. C.
Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase
from the Gram-Positive Bacterial Pathogen A. vaginae, an
Immunoevasive Factor that Interacts with the Human C5a
Anaphylatoxin. Front. Microbiol. 2017, 8, 541.
(64) Hol, W. G.; Halie, L. M.; Sander, C. Dipoles of the α-helix and
β-sheet: their role in protein folding. Nature 1981, 294, 532−536.
(65) Song, R.; Wu, X.; Xue, B.; Yang, Y.; Huang, W.; Zeng, G.;
Wang, J.; Li, W.; Cao, Y.; Wang, W.; et al. Principles governing
catalytic activity of self-assembled short peptides. J. Am. Chem. Soc.
2019, 141, 223−231.
(66) Zhang, C.; Shafi, R.; Lampel, A.; MacPherson, D.; Pappas, C.
G.; Narang, V.; Wang, T.; Maldarelli, C.; Ulijn, R. V. Switchable
hydrolase based on reversible formation of supramolecular catalytic
site using a self-assembling peptide. Angew. Chem., Int. Ed. 2017, 56,
14511−14515.
(67) Zhang, C.; Xue, X.; Luo, Q.; Li, Y.; Yang, K.; Zhuang, X.; Jiang,
Y.; Zhang, J.; Liu, J.; Zou, G.; Liang, X.-J. Self-Assembled Peptide
Nanofibers Designed as Biological Enzymes for Catalyzing Ester
Hydrolysis. ACS Nano 2014, 8, 11715−11723.
(68) Rink, W. M.; Thomas, F. De Novo Designed α-Helical Coiled-
Coil Peptides as Scaffolds for Chemical Reactions. Chem. − Eur. J.
2019, 25, 1665−1677.
(69) Lombardi, A.; Pirro, F.; Maglio, O.; Chino, M.; DeGrado, W. F.
De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold,
Diverse Reactivities. Acc. Chem. Res. 2019, 52, 1148−1159.
(70) Bisswanger, H. From Reaction Order to the Michaelis−Menten
Law: Fundamental Relationships of Enzyme Kinetics. In Enzyme
Kinetics; John Wiley & Sons, Inc., 2017; pp 55−86.
(51) Wang, M.; Lv, Y.; Liu, X.; Qi, W.; Su, R.; He, Z. Enhancing the
activity of peptide-based artificial hydrolase with catalytic Ser/His/
Asp triad and molecular imprinting. ACS Appl. Mater. Interfaces 2016,
8, 14133−14141.
(71) Lineweaver, H.; Burk, D. The determination of enzyme
dissociation constants. J. Am. Chem. Soc. 1934, 56, 658−666.
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ACS Catal. 2021, 11, 5839−5849