Copper-Containing Catalytic Amyloids Promote Phosphoester Hydrolysis and Tandem Reactions

Article abstract of DOI:10.1021/acscatal.7b03323

Self-assembly of short de novo designed peptides gives rise to catalytic amyloids capable of facilitating multiple chemical transformations. We show that catalytic amyloids can efficiently hydrolyze paraoxon, which is a widely used, highly toxic organopho

Full text of DOI:10.1021/acscatal.7b03323

Subscriber access provided by READING UNIV
Letter
Copper-Containing Catalytic Amyloids Promote
Phosphoester Hydrolysis and Tandem Reactions
Zsofia Lengyel, Caroline M. Rufo, Yurii S. Moroz, Olga V. Makhlynets, and Ivan V Korendovych
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03323 • Publication Date (Web): 22 Nov 2017
Downloaded from http://pubs.acs.org on November 22, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
ACS Catalysis
1
2
3
4
5
6
7
8
9
Copper-Containing Catalytic Amyloids Promote Phosphoester Hy-
drolysis and Tandem Reactions
Zsófia Lengyel,† Caroline M. Rufo, † Yurii S. Moroz, †,‡ Olga V. Makhlynets, † Ivan V.
Korendovych†,*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Department of Chemistry, Syracuse University, 111 College Place, Syracuse, NY 13244.
ABSTRACT: Self-assembly of short de novo designed peptides gives rise to catalytic amyloids capable of facilitating mul-
tiple chemical transformations. We show that catalytic amyloids can efficiently hydrolyze paraoxon, a widely used, highly
toxic organophosphate pesticide. Moreover, these robust and inexpensive metal-containing materials can be easily depos-
ited on various surfaces producing catalytic flow devices. Finally, functional promiscuity of catalytic amyloids promotes
tandem hydrolysis/oxidation reactions. High efficiency discovered in a very small library of peptides suggests an enor-
mous potential for further improvement of catalytic properties both in terms of catalytic efficiency and substrate scope.
KEYWORDS: cascade reaction, self-assembly, peptides, copper, paraoxon
Self-assembly of short peptides presents a facile method
of producing a large number of extremely functionally
and structurally diverse amyloid-like assemblies.1-11 Re-
cently we have shown that self-assembly of de novo de-
signed peptides in the presence of metal ions produces
catalytic amyloids capable of efficient catalysts of a num-
ber of hydrolytic and redox chemical transformations.12-14
The catalytic efficiency of these nanoassemblies in model
reactions is higher than that of some enzymes by weight
and can be further improved by synergistic interactions
observed in mixtures of peptides with different sequences.
The resulting fibrils are extremely homogeneous, can
function under high pressure,15 and exhibit moderate sub-
strate preference.16 Moreover, catalytic metalloamyloids
with diverse sets of primary sequences17-19 and metal ions20
have been reported.
Given the exciting possibilities in catalysis provided by
amyloid assemblies we aimed to embark beyond simple
model substrates and determine whether catalytic amy-
loids can be successfully employed to hydrolyze paraoxon,
a widely used, highly toxic organophosphate pesticide
(Scheme 1), that contributes to estimated three million
organophosphate poisonings a year worldwide.21 Moreo-
Scheme 1. Hydrolysis of paraoxon and tandem reac-
ver, chemical similarity of paraoxon to toxic nerve agents,
makes it a useful test system to develop efficient and
practical strategies for chemical weapons remediation.
Paraoxon is a challenging substrate for hydrolysis (back-
ground hydrolysis kuncat = 7.97 × 10-7 min-1 at pH 7.8),22
thus this pesticide persists in the environment for a pro-
longed period of time. While a number of highly efficient
paraoxonases have been already developed using directed
evolution of related proteins,23 catalytic amyloids offer a
number of practical advantages such as robustness, low
cost and ease of modification.
tions catalyzed by catalytic amyloids.
Hydrolysis of paraoxon is often facilitated by metalloco-
factors, CuII being one of the most reactive ones.24 Given
the established ability of catalytic amyloids to
ACS Paragon Plus Environment

ACS Catalysis
Page 2 of 5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. 7IY forms amyloid-like fibrils that catalyze paraoxon hydrolysis. a) AFM images of the fibrils formed by 7IY in the pres-
ence of CuII; peptides were incubated for 15-30 min prior to deposition onto mica; b) CD spectra of 7IY under various conditions
([7IY] = [CuII]= 25 µM, 5 mM HEPES, pH 8.0); c) dependence of initial rate of paraoxon hydrolysis catalyzed by 7IY on the sub-
strate concentration; peptides were incubated at 37 °C for 30 min prior to addition of substrate to ensure fibril formation.
bind this metal ion13-14 we have tested the ability of sev-
eral previously synthesized peptides to catalyze paraoxon
hydrolysis in the presence of copper.
lysts.30-32 It compares well to many polymer-based hetero-
geneous catalysts albeit it is difficult to draw direct com-
parison as experimental conditions vary widely (Table S1,
Supporting Information).
The results of the screening (Fig. S1, Supporting Infor-
mation) show that catalytic amyloids can provide sub-
stantial acceleration of paraoxon hydrolysis over the
background rate. Peptide 7IY (Ac-IHIHIYI-NH2) showed
very high activity in the initial screen, thus we chose it for
in depth characterization. Interestingly, several peptides
with very minor differences in primary sequences as com-
pared to 7IY showed activity that is somewhat lower than
that of free copper ions, consistent with unproductive
binding of the metal ion to peptide.
7IY reaches its maximum activity at ca. 1 equiv of cop-
per (Fig. S5, Supporting Information). EPR spectrum of
CuII-bound 7IY shows a typical Type II copper similar to
the one observed in other catalytic amyloid peptides (Fig.
S6, Supporting Information). The g||, g⊥, and A|| parame-
ters are very similar to the ones observed for 7IQ suggest-
ing nearly identical metal coordination sphere.13 The lack
of a charge transfer band corresponding to copper-
phenolate interaction suggests that Tyr6 is not directly
involved in copper coordination.
7IY forms amyloid-like fibrils in the presence of copper
as evidenced by atomic force microscopy (AFM), circular
dichroism (CD) and thioflavin T (ThT) fluorescence data
(Fig. 1a-b, Fig. S2, Supporting Information). Interestingly
ThT fluorescence in the presence of copper fibrils is much
diminished when compared to fibrils that do not contain
the metal. This is consistent with fluorescence quenching
by paramagnetic copper ions. The fibrils show well-
defined block morphology, with most probable thickness
of ~2.5 Å consistent with a single bilayer of cross-beta
oriented peptides (Fig. S3, Supporting Information) in
agreement with previous SAXS18 and ssNMR25 studies.
Formation of the fibrils is quite rapid – incubation of
the peptides for 24 hours does not result in significant
additional fibrillization (Fig. S2, Supporting Information).
The dependence of the initial rate of hydrolysis as a func-
tion of substrate concentration (Fig. 1c) can be fit to the
Michaelis-Menten equation to yield kinetic parameters of
Figure 2. Hydrolysis of paraoxon facilitated by multiple
passes of the substrate solution through a flow device at 1
mL/min (a) prepared by a deposition of copper-containing
fibrils on PES support (b).
The comparison of the rates of paraoxon hydrolysis
with those of the p-nitrophenylacetate (pNPA) is instruc-
tive. Consistent with the previous study18 7IY shows very
high efficiency in hydrolyzing pNPA in the presence of
zinc (kcat/KM = 5888 ± 195 M-1min-1). However, the rate of
7IY-promoted pNPA hydrolysis in the presence of copper,
while still quite significant, is much slower (kcat/KM = 806
± 100 M-1min-1). The reverse trend is true for phosphoe-
k
cat = 4.8 ± 0.9 × 10-3 min-1, KM = 2.9 ± 0.8 mM and kcat/KM
= 1.7 ± 0.6 M-1min-1. The pH profile of the activity (Fig. S4,
Supporting Information) is consistent with a single water
deprotonation step with an effective pKa of 8.3. While the
resulting kcat/kuncat ratio of 3.5 × 103 at pH 8.0 is inferior to
those observed for engineered enzymes,23, 26-28 it is on par
29
with catalytic antibodies22,
and small molecule cata-
ACS Paragon Plus Environment

Page 3 of 5
ACS Catalysis
sters as copper-containing fibrils are more efficient in
measured the rate of oxidation of DCFH as well as the
rate of hydrolysis of DCF-DA, a substitute for DCFH-DA
as hydrolysis of DCFH-DA does not produce a convenient
fluoro- or chromophore (Scheme 1).
1
2
3
4
5
6
7
8
paraoxon hydrolysis (Fig. S7, Supporting Information).
Catalytic amyloids being heterogeneous catalysts pro-
vide a very significant degree of molecular control akin to
that found in homogeneous catalysts. In order to explore
practicality of use of catalytic amyloids we have created a
flow catalyst. Catalytic amyloids prepared by addition of
copper ions to 7IY were filtered through a 0.22 µm poly-
ethersulfone (PES) filter (Fig. 2). Once amyloids are
formed and filtered through the PES support, very little
peptide was observed in the filtrate (Fig. S8, Supporting
Information). The resulting filter can be immediately
used as a heterogeneous catalyst for hydrolysis of
paraoxon in a flow system (Fig. 2). While the overall activ-
ity of the amyloids deposited on the filter is somewhat
diminished as compared to the original suspension, mul-
tiple passes of the substrate solution can be done to facili-
tate paraoxon hydrolysis in a continuous manner. The
simplicity of the catalyst deposition onto unfunctional-
ized filter support will greatly aid in practical applications
that require easy catalyst separation and recycling.
The rate of the hydrolysis step is about 10-fold slower
than the rate of oxidation in both cases, making it rate
limiting. Interestingly, the rate of DCF-DA hydrolysis is
about 2-fold lower than the overall rate of DCHF-DA con-
version to DCF, meaning that the combined reaction rate
is faster than the rate-limiting step.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Kinetic parameters for hydrolysis and/or
oxidation of various fluorescein derivatives.
Substrate
DCFH-DA
DCFH-DA
DCFH
Product
DCF
Catalyst
7IY
k2, M-1 min-1
61.2 ± 9.6
DCF
7IQ
97.2 ± 6.6
288.6 ± 20.4
291.0 ± 23.4
33.6 ± 3
DCF
7IY
DCFH
DCF
7IQ
DCF-DA
DCF-DA
DCF
7IY
DCF
7IQ
30.0 ± 0.6
Encouraged by high chemical versatility of catalytic
amyloids we set out to explore whether they can simulta-
neously promote multiple reactions at the same time.
High hydrolytic activity of copper-containing catalytic
amyloids together with their ability to activate oxygen13
prompted us to explore whether they can facilitate cas-
cade reactions that include hydrolytic and redox compo-
nents. Cascade (also referred to as tandem) reactions are
multi-step sequential chemical transformations that re-
quire formation of the first product for the initiation of
the next step.33 As a test substrate for cascade reactions
we have chosen a well-established substrate - 2’,7’-
dichlorofluorescin diacetate (DCFH-DA) that normally
undergoes hydrolysis to produce 2’,7’-dichlorofluorescin
(DCFH), which is in turn gets oxidized to yield highly
fluorescent 2’,7’-dichlorofluorescein (DCF), Scheme 1.34
Alternatively, DCFH-DA can get first oxidized to DCF-DA
followed by hydrolysis to produce DCF, although the
overall rate of this path is slower.35 The formation of
product can be followed by fluorescence and/or appear-
ance of the absorption band at 504 nm (Fig. S9, Support-
ing Information). The heterogeneous nature of copper-
containing amyloids is expected to provide an additional
kinetic advantage – the product of hydrolysis can associ-
ate with the catalytic amyloids for some degree (as evi-
denced by relatively low KM values observed for a variety
of substrates), thus creating a higher effective concentra-
tion for the oxidation step giving an overall boost in the
observed kinetics.
This can be explained by either contribution of the al-
ternative path (oxidation followed by hydrolysis) to the
increase of overall rate or by a difference in the hydrolysis
rate of DCF-DA compared to that of DCFH-DA. In order
to test these hypotheses we tested the reactivity of a
closely related peptide 7IQ (Ac-IHIHIQI-NH2), that has
previously shown superior activity in redox catalysis,13 in
DCFH-DA hydrolysis and oxidation. Despite very similar
activities of 7IQ and 7IY in DCF-DA hydrolysis and DCFH
oxidation, the overall conversion of DCFH-DA to DCF is
almost 2-fold higher for 7IQ (97 ± 7 M-1 min-1) than that of
7IY. This observation essentially eliminates the possibility
that DCF-DA is a poor substitute for DCFH-DA for kinetic
studies and is consistent with multiple pathways for oxi-
dation and hydrolysis simultaneously available for catalyt-
ic amyloids that result in the overall rate acceleration.
In summary, we have shown that catalytic amyloids are
capable of facilitating hydrolysis of paraoxon, a challeng-
ing substrate of practical importance, by more than three
orders of magnitude (kcat/kuncat). We have also demon-
strated that catalytic amyloids can be easily utilized in
flow cells. We have shown for the first time that promis-
cuity of catalytic amyloids allows them to facilitate tan-
dem transformations. High activity of catalytic amyloids
seen in a very limited library of short peptides suggests an
incredible potential for further improvement of catalytic
properties both in terms of catalytic efficiency and sub-
strate scope.
We have tested ability of 7IY to catalyze hydrolysis and
oxidation of DCFH-DA. The results of kinetic experiments
are shown in Table 1 and Fig. S10, Supporting Information.
7IY simultaneously catalyzes hydrolysis and oxidation of
DCFH-DA in buffer at pH 8.0 with the effective second
order rate constants of 61 ± 9 M-1 min-1; the background
rate of the reaction under these conditions is essentially
negligible (Table S2, Supporting Information). In order to
gain mechanistic insight into this transformation we have
ASSOCIATED CONTENT
Supporting Information. Details of peptide synthesis,
characterization, and kinetic studies. The Supporting Infor-
mation is available free of charge on the ACS Publications
website.
AUTHOR INFORMATION
Corresponding Author
ACS Paragon Plus Environment

ACS Catalysis
Page 4 of 5
18.
Al-Garawi, Z.S.; McIntosh, B. A.; Neill-Hall, D.;
ikorendo@syr.edu.
Hatimy, A. A.; Sweet, S. M.; Bagley, M. C.; Serpell, L. C.
Nanoscale 2017, 9, 10773-10783.
1
2
3
4
5
6
7
8
Present Addresses
‡Department of Chemistry, Taras Shevchenko National Uni-
versity of Kyiv, 64 Volodymyrska St., Kyiv 01601, Ukraine.
19.
Zhang, C. Q.; Xue, X. D.; Luo, Q.; Li, Y. W.; Yang, K. N.;
Zhuang, X. X.; Jiang, Y. G.; Zhang, J. C.; Liu, J. Q.; Zou, G. Z.;
Liang, X. J. ACS Nano 2014, 8, 11715-11723.
20.
Biophys. Res. Commun. 2017, 482, 1194-1200.
21. Word Health Organization., Public health impact of
pesticides used in agriculture. 1990.
Monasterio, O.; Nova, E.; Diaz-Espinoza, R. Biochem.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
22.
7636.
23.
Lavey, B. J.; Janda, K. D. J. Org. Chem. 1996, 61, 7633-
9
Funding Sources
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Goldsmith, M.; Ashani, Y.; Margalit, R.; Nyska, A.;
No competing financial interests have been declared. This
work was supported by the NIH (grant GM119634 to I.V.K.),
the NSF (grant 1332349 to I.V.K.) and the Alexander von
Humboldt Foundation. The EPR facilities at Cornell (ACERT)
are supported by an NIH grant P41GM103521.
Mirelman, D.; Tawfik, D. S. Chem. Biol. Interact. 2016, 259, 242-
251.
24.
Mortland, M. M.; Raman, K. V. J. Agric. Food Chem.
1967, 15, 163-167.
25.
Lee, M.; Wang, T.; Makhlynets, O. V.; Wu, Y.; Polizzi,
N. F.; Wu, H.; Gosavi, P. M.; Stöhr, J.; Korendovych, I. V.;
DeGrado, W. F.; Hong, M., Proc. Natl. Acad. Sci. U.S.A. 2017, 114,
6191-6196.
ACKNOWLEDGMENT
AFM images were collected at the Argonne National Lab
(Center for Nanoscale Materials). We thank Dr. Boris
Dzikovski for assistance with EPR experiments.
26.
Cerasoli, D. M. Chem. Biol. Interact. 2013, 203, 181-185.
27. Xiang, D. F.; Kolb, P.; Fedorov, A. A.; Meier, M. M.;
Kirby, S. D.; Norris, J. R.; Smith, J. R.; Bahnson, B. J.;
Fedorov, L. V.; Nguyen, T. T.; Sterner, R.; Almo, S. C.; Shoichet,
B. K.; Raushel, F. M. Biochemistry 2009, 48, 2237-2247.
REFERENCES
28.
Ugwumba, I. N.; Ozawa, K.; Xu, Z.-Q.; Ely, F.; Foo, J.-
1.
Duncan, K. L.; Ulijn, R. V. Biocatalysis 2015, 1, 67-81.
L.; Herlt, A. J.; Coppin, C.; Brown, S.; Taylor, M. C.; Ollis, D. L.;
Mander, L. N.; Schenk, G.; Dixon, N. E.; Otting, G.; Oakeshott, J.
G.; Jackson, C. J. J. Am. Chem. Soc 2011, 133, 326-333.
2.
Maeda, Y.; Makhlynets, O. V.; Matsui, H.;
Korendovych, I. V. Annu. Rev. Biomed. Eng. 2016, 18, 311-328.
3. Branco, M. C.; Sigano, D. M.; Schneider, J. P. Curr.
Opin. Chem. Biol. 2011, 15, 427-434.
29.
K. D. Bioorg. Med. Chem. Lett. 2005, 15, 4304-4307.
30. Kennedy, D. J.; Mayer, B. P.; Baker, S. E.; Valdez, C. A.
Inorg. Chim. Acta 2015, 436, 123-131.
Xu, Y.; Yamamoto, N.; Ruiz, D. I.; Kubitz, D. S.; Janda,
4.
Omosun, T. O.; Hsieh, M.-C.; Childers, W. S.; Das, D.;
Mehta, A. K.; Anthony, N. R.; Pan, T.; Grover, M. A.; Berland, K.
M.; Lynn, D. G. Nat. Chem. 2017, 9, 805-809
31.
2330-2333.
32.
2106.
33.
Morrow, J. R.; Trogler, W. C. Inorg. Chem. 1989, 28,
5.
Swanekamp, R. J.; Welch, J. J.; Nilsson, B. L. Chem.
Commun. 2014, 50, 10133-10136.
Kuo, L. Y.; Perera, N. M. Inorg. Chem. 2000, 39, 2103-
Filice, M.; Palomo, J. M. ACS Catalysis 2014, 4, 1588-
LeBel, C. P.; Ischiropoulos, H.; Bondy, S. G. Chem. Res.
6.
Liyanage, W.; Brennessel, W. W.; Nilsson, B. L.
Langmuir 2015, 31, 9933-9942.
7.
Fry, H. C.; Garcia, J. M.; Medina, M. J.; Ricoy, U. M.;
1598.
34.
Gosztola, D. J.; Nikiforov, M. P.; Palmer, L. C.; Stupp, S. I. J. Am.
Chem. Soc. 2012, 134, 14646-14649.
Toxicol. 1992, 5, 227-231.
8.
Solomon, L. A.; Kronenberg, J. B.; Fry, H. C. J. Am.
35.
Bonini, M. G.; Rota, C.; Tomasi, A.; Mason, R. P. Free
Chem. Soc. 2017, 139, 8497-8507.
Radic. Biol. Med. 2006, 40, 968-975.
9.
Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady,
R. L.; Woolfson, D. N. Nat. Chem. 2016, 8, 837-844.
10.
Escuder, B. Chem. – Eur. J. 2017, 23, 981-993.
11. Rubinov, B.; Wagner, N.; Rapaport, H.; Ashkenasy, G.
Angew. Chem. Int. Ed. 2009, 48, 6683-6686.
12. Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stöhr, J.;
Singh, N.; Kumar, M.; Miravet, J. F.; Ulijn, R. V.;
Smith, T. A.; Hu, X.; DeGrado, W. F.; Korendovych, I. V. Nat.
Chem. 2014, 6, 303-309.
13.
Angew. Chem. Int. Ed.. 2016, 55, 9017-9020.
14. Sternisha, A.; Makhlynets, O. In Synthetic Protein
Makhlynets, O. V.; Gosavi, P. M.; Korendovych, I. V.
Switches: Methods and Protocols, Stein, V., Ed. Springer New
York: New York, 2017; p 59-68.
15.
Luong, T. Q.; Erwin, N.; Neumann, M.; Schmidt, A.;
Loos, C.; Schmidt, V.; Fändrich, M.; Winter, R. Angew. Chem. Int.
Ed. 2016, 55, 12412-12416.
16.
Heier, J. L.; Mikolajczak, D. J.; Böttcher, C.; Koksch, B.
Pept. Sci. 2017, 108, e23003.
17.
Friedmann, M. P.; Torbeev, V.; Zelenay, V.; Sobol, A.;
Greenwald, J.; Riek, R. PLoS ONE 2015, 10, e0143948.
ACS Paragon Plus Environment

Page 5 of 5
ACS Catalysis
Table of Contents (TOC)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment