Substrate specificity of an actively assembling amyloid catalyst

Article abstract of DOI:10.1002/bip.23003

In the presence of Zn²⁺, the catalytic, amyloid-forming peptide Ac-IHIHIQI-NH₂, was found to exhibit enhanced selectivity for hydrophobic p-nitrophenyl ester substrates while in the process of self-assembly. As opposed to the substrate p-nitrophenyl acetate, which was more effectively hydrolyzed with Ac-IHIHIQI-NH₂ in its fully fibrillar state, the hydrophobic substrate Z-L-Phe-ONp was converted with a second-order rate constant more than 11-times greater when the catalyst was actively assembling. Under such conditions, Z-L-Phe-ONp hydrolysis proceeded at a greater velocity than the more hydrophilic and otherwise more labile ester Boc-L-Asn-ONp. When assembling, the catalyst also showed increased selectivity for the L-enantiomer of Z-Phe-ONp. These findings suggest the occurrence of increased interactions of hydrophobic moieties of the substrate with exposed hydrophobic surfaces of the assembling peptides and present valuable features for future de novo design consideration.

Full text of DOI:10.1002/bip.23003

Substrate specificity of an actively assembling amyloid catalyst
Jason L. Heier, Dorian J. Mikolajczak, Christoph Böttcher and Beate Koksch
Introduction
[1]
Designing a protein de novo to exhibit the natureꢀhoned properties of an enzyme can be a
disheartening task. Such efforts are not only made with intentions to construct enzymes, but also
to study the relationship between protein folding and enzymatic function. To gain a clearer
understanding of residue behavior in a proteinꢀlike periphery, a minimalist or “bottomꢀup”
[2]
approach to de novo design can be taken. In doing so, peptides, particularly those which selfꢀ
assemble into predictable, proteinꢀlike structures, provide a valuable platform for protein
engineering. The use of such peptides in the bottomꢀup approach, therefore, offers a higher
resolution of the interactions vital not only for catalytic activity, but also substrate specificity.
[2c,3]
Initial kinetic experiments performed on hydrolytic de novoꢀdesigned protein catalysts
or
rationally redesigned enzymes often employ the substrate pꢀnitrophenyl acetate (pNPA)
Fig.1). As a model ester substrate, pNPA meets the most rudimentary requirements, an acetyl
[4]
(
acyl component and the standard nitrophenolate (pKa = 7) as chromophore used to monitor
reaction progress. Because pNPA is achiral and lacks a significant functional group or
hydrophobic portion, it is unlikely to participate in any specific interaction with the catalyst. In
other words, pNPA is a suitable substrate for evaluating general catalytic performance, but offers
little information regarding specificity.
[
3a]
Recently, Rufo and coworkers reported the fibrilꢀforming peptide AcꢀIHIHIQIꢀNH , which was
2
2+
shown to efficiently catalyze pNPA hydrolysis in the presence of Zn . The de novoꢀdesigned Acꢀ
IHIHIQIꢀNH is believed to selfꢀassemble into a parallel βꢀsheet which allows Zn to be
2+
2
coordinated by three histidine residues leaving an open site for water. This carbonic anhydraseꢀ
2
+
[5]
like His Zn active site affects hydrolysis by employing the zincꢀbound hydroxide nucleophile
3
[5c]
2+
(
ZHN) mechanism. In kinetic experiments, AcꢀIHIHIQIꢀNH with Zn was found to catalyze
2
ꢀ1 ꢀ1
pNPA hydrolysis with a k /KM of 62 M s at pH 8 and a secondꢀorder rate constant
approaching a respectable 360 M s under slightly more basic conditions.
cat
ꢀ1
ꢀ1
[3a]
The catalytic activity of AcꢀIHIHIQIꢀNH coupled with its interesting structural aspects and
2
aggregation behavior raised further interest regarding its substrate specificity. In the work
presented here, Lꢀ and Dꢀenantiomers of substrates Nꢀbenzyloxycarbonylꢀprotected
phenylalanine pꢀnitrophenyl ester (ZꢀPheꢀONp) (Fig. 1) and the less hydrophobic Nꢀ
butyloxycarbonylꢀprotectedꢀasparagine pꢀnitrophenyl ester (BocꢀAsnꢀONp) were applied to study
the impact hydrophobic interactions have on the catalytic activity and substrate specificity of
fibrilꢀforming AcꢀIHIHIQIꢀNH . To this end, kinetic experiments were undertaken in which ester
2
substrates were presented to AcꢀIHIHIQIꢀNH either in its fullyꢀfibrillar form following preꢀ
2
2+
o
incubation with Zn at 20 C, or while in the process of fibril assembly. In this study, zinc was
used to increase the catalytic activity of AcꢀIHIHIQIꢀNH and was not required to achieve
2
amyloid fibril formation as shown by thioflavin T (ThT) binding (Fig. S7).
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Page 2 of 15
Results and Discussion
Prior to performing kinetic studies, a stock solution of AcꢀIHIHIQIꢀNH at pH 2 was prepared.
2
Under such acidic conditions, no secondary protein structure was observed by CD spectroscopy
2+
(
Figures 2 and S3). However, when added to pH 7.5 buffer containing Zn as well as the ester
substrates (Figures S4 – S6), an intense minimum (207 nm) in ellipticity quickly developed. This
[3a]
finding corresponds to the results of similar studies reported by Rufo et al. and CD spectra of
[6]
other βꢀsheet, amyloidꢀforming peptides.
Since the substrates employed in this kinetic study vary in their hydrophobic nature with ZꢀPheꢀ
ONp being only sparingly soluble in water, low substrate concentrations were used. Therefore,
[7]
pseudoꢀfirst order kinetics as opposed to saturation kinetics were implemented. As expected,
2+
hydrolysis of pNPA occurred most rapidly when treated with fibrils incubated with Zn 24 hours
prior to the reaction (Figures 3 and S13). The secondꢀorder rate constant k at pH 7.5 was
2
ꢀ1 ꢀ1
determined to be 87 M s (Table 1) based on previous findings in which two peptides constitute
[
3a]
one active site. On the other hand, k corresponding to the actively assembling catalyst was
2
ꢀ
1 ꢀ1
just over 40 M s . The faster hydrolysis of pNPA observed in the presence of the preꢀincubated
fibrils was likely due to the larger number of established active sites as opposed to peptides still
in the process of assembly and aggregation.
Remarkably, the opposite effect was observed when ZꢀLꢀPheꢀONp was employed as substrate.
In this case, AcꢀIHIHIQIꢀNH was most active when selfꢀassembly and the reaction occurred
2
simultaneously (Fig. 4). The secondꢀorder rate constant of ZꢀLꢀPheꢀONp hydrolysis mediated by
4
ꢀ1 ꢀ1
the actively assembling catalyst was found to be 2.2 x 10 M s , more than 11ꢀfold greater than
the rate constant corresponding to preꢀincubated AcꢀIHIHIQIꢀNH fibrils (Table 1). Furthermore,
2
the turnover rate (apparent firstꢀorder rate constant) was more than 70ꢀfold greater with actively
assembling AcꢀIHIHIQIꢀNH present than when uncatalyzed. In addition cryoꢀTEM studies were
2
performed revealing immediate fibril formation in the presence of ZꢀLꢀPheꢀONp just 25 seconds
after the initiation of the reaction with activelyꢀassembling AcꢀIHIHIQIꢀNH (Figures 5 and S7).
2
This finding is likely the result of increased interactions between hydrophobic moieties of the
substrate and exposed hydrophobic regions of the assembling catalyst (Fig. 6). Although the
exact mode of substrate/peptide catalyst interaction is not clearly understood, it seems probable
that the extremely hydrophobic ZꢀPheꢀONp is drawn into close proximity to the hydrophobic
[
7f,8]
regions of the catalyst as has often been observed in micellar enzyme mimics
enzymes
and artificial
[7a,c]
[7e]
. It may even be possible for ZꢀPheꢀONp to intercalate within the βꢀsheets of Acꢀ
IHIHIQIꢀNH during assembly. Therefore, as aggregation progresses the substrate may already
2
be present in the vicinity of the newly established active site. In the case of a fullyꢀformed fibril
catalyst, such hydrophobic interactions are less likely to take place as the hydrophobic
isoleucine residues are well buried prior to the approach of the substrate. Heightened catalytic
efficiency during selfꢀassembly was not observed when pNPA was employed as a substrate
since it is comparatively less hydrophobic. In addition, the possible involvement of piꢀ
[9]
interactions or steric effects occurring between substrate and catalyst should not be
overlooked. The presence of three aromatic groups make ZꢀPheꢀONp more likely than pNPA to
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2+
participate in piꢀpi interactions with histidine residues or cationꢀpi interactions with Zn of the
2+
catalyst. Such piꢀinteractions of substrate with catalyst would occur differently prior to Zn
2+
2+
coordination (i.e. during selfꢀassembly with Zn ) as opposed to when Zn is already
coordinated in the preꢀincubated fibrillar form. Furthermore, with respect to steric effects, the
bulky benzyloxycarbonyl group and benzyl sideꢀchain of phenylalanine may not allow the ZꢀPheꢀ
ONp substrate to approach the broad surface of fullyꢀformed fibrils as easily as it would smaller
assemblies.
Compared to ZꢀLꢀPheꢀONp, which was quickly cleaved by the selfꢀassembling form of Acꢀ
IHIHIQIꢀNH , hydrolysis of the more hydrophilic BocꢀLꢀAsnꢀONp occurred less rapidly (Table 1).
2
Although the more labile ester BocꢀLꢀAsnꢀONp undergoes hydrolysis more rapidly than ZꢀLꢀPheꢀ
ONp in the absence of catalyst, kinetic studies showed that AcꢀIHIHIQIꢀNH , while selfꢀ
2
2+
assembling in the presence of Zn , was clearly selective for the more hydrophobic ZꢀLꢀPheꢀ
ONp (Fig. 7a). The ability of AcꢀIHIHIQIꢀNH to exhaust the hydrophobic ZꢀLꢀPheꢀONp more
2
than fiveꢀfold faster than the more reactive, yet more hydrophilic BocꢀLꢀAsnꢀONp suggests that
the hydrophobic nature of the substrate and its interactions with the catalyst play a discernible
role in the selectivity of the selfꢀassembling catalytic peptide. This conclusion is also supported
by the observation that selectivity for ZꢀLꢀPheꢀONp over BocꢀLꢀAsnꢀONp was found to be greatly
2+
diminished when the catalyst was incubated with Zn prior to the initiation of the reaction (Fig.
b).
7
In addition to substrate selectivity, both forms of AcꢀIHIHIQIꢀNH2 were tested for
enantioselectivity using ZꢀDꢀPheꢀONp and BocꢀDꢀAsnꢀONp as substrates (see Supporting
2+
Information). While actively assembling, AcꢀIHIHIQIꢀNH in the presence of Zn was found to be
2
selective for the Lꢀenantiomer of both substrates (Figures S3 and S4). The ratio of secondꢀorder
rate constants (k :k ) resulted in enantioselectivity values (E ) of 3.0 and 2.5 corresponding to
2L
2D
L/D
ZꢀPheꢀONp and BocꢀAsnꢀONp, respectively (Table 1). However, enantioselectivity for ZꢀLꢀPheꢀ
2+
ONp (E ) was greatly reduced, to 1.1, when AcꢀIHIHIQIꢀNH was incubated with Zn prior to
L/D
2
use (Fig. S5). To a lesser extent, a reduction in enantioselectivity for BocꢀLꢀAsnꢀONp (EL/D = 1.7)
was also observed (Fig. S6). Whether in the process of selfꢀassembly or fibrillar, the form of Acꢀ
IHIHIQIꢀNH , which is to a great extent directed by its hydrophobic nature, has a lesser effect in
2
distinguishing between the more hydrophilic BocꢀAsnꢀONp enantiomers than those of ZꢀPheꢀ
ONp.
Besides its catalytic capability, the simple βꢀsheet structure of AcꢀIHIHIQIꢀNH provides a
2
reasonably selective catalyst when properly applied. The most encountered limitation of βꢀsheet
structures in de novo design is solubility. The results shown here are suggestive of what is
possible, especially in terms of substrate selectivity, if such structures could be stabilized in
[1c]
solution as has been postulated in the literature. Future efforts to stabilize sequences similar
to IHIHIQI between αꢀhelices could help define a stable, threeꢀdimensional active site enhanced
by secondary interactions and additional features contributing to selectivity.
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Materials and Methods
Peptide synthesis: Peptide AcꢀIHIHIQIꢀNH2 was manually synthesized using standard
[10]
fluorenylmethyloxycarbonyl (Fmoc) methodology on Rink Amide MBHA resin. The coupling
procedure of each Fmocꢀprotected amino acid (5 eq.) was performed twice employing reagents
N,N’ꢀdiisoproplycarbodiimide (DIC) (4.9 eq.) and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAt) (5 eq.) in
DMF. After coupling and Fmoc deprotection of the Nꢀterminal isoleucine, the aminoꢀgroup was
acetylated with a solution containing acetic anhydride (10%) and diisopropylethylamine (10%) in
DMF. Sideꢀchain deprotection and peptide/resin cleavage was accomplished in a cocktail of
trifluoroacetic acid (TFA), H O and triisopropylsilane (TIS) (95:2:3, v/v). Crude AcꢀIHIHIQIꢀNH
2
2
was precipitated in iceꢀcold diethyl ether and purified via reverseꢀphase HPLC (Knauer Smartline
000) with preparative column Jupiter C4 from Phenomenex applying a linear gradient of solvent
5
A (70% H O, 30% MeCN, 0.1% TFA) and solvent B (MeCN with 0.1% TFA). The identity of Acꢀ
2
IHIHIQIꢀNH was confirmed by ESIꢀToF mass spectrometry (Agilent 6210; Fig. S1) and its purity
2
was further assessed with analytical RPꢀHPLC (Elite LaChrome from VWRꢀHitachi with the
column Kinetix C18 (250 x 4.6 mm) from Phenomenex; Fig. S2).
Ac-IHIHIQI-NH stock solution preparation: Lyophilized peptide was dissolved in 10 mM HCl.
2
To determine the concentration of this solution a 10 ꢁL aliquot was withdrawn, diluted 100ꢀfold
with 10 mM HCl and its absorbance at 214 nM was measured. The concentration of the solution
ꢀ1
ꢀ1
was calculated using a molar extinction coefficient of 17,956 Lmol cm acquired according to
[11]
Kuipers and Gruppen.
The peptide solution was diluted accordingly with 10 mM HCl to
achieve a pH 2 stock solution of 1.1 mM.
Kinetic assays: Ester hydrolysis reactions were performed in 1 cm quartz cuvettes (3.5 mL;
Hellma Analytics) equipped with a Teflon stopper. All reactions with exception of those used to
monitor background hydrolysis were performed with 10 ꢁM AcꢀIHIHIQIꢀNH , which corresponds
2
[3a]
o
to 5 ꢁM of the characterized catalyst. Reactions took place at 20 C in 50 mM Tris/HCl buffer
pH 7.5 with 100 ꢁM ZnCl . Assays employing pNPA used an initial substrate concentration of 50
2
ꢁ
M. Reactions using substrates ZꢀPheꢀONp or BocꢀAsnꢀONp were carried out with a starting
concentration of 10 ꢁM. Reaction progress was monitored by measuring the formation of pꢀ
nitrophenolate at 405 nm as a function of time (A ) using a thermostatꢀequipped UVꢀVis
t
spectrophotometer (Cary 50 Bio, Varion). Upon completion of the reactions (>24 h), final
absorbance (A ) measurements were taken at 405 nm. Apparent firstꢀorder rate constants (k )
∞
1
were determined as the negative slope of the firstꢀorder plot of the linear Equation 1.
Ln (A ꢀ A ) = ꢀk t + Ln A
∞
(Equation 1)
∞
t
1
Secondꢀorder rate constants (k ) were obtained by subtracting the apparent firstꢀorder rate
2
constant corresponding to the background reaction (k ) from the apparent firstꢀorder constants
1b
determined in the presence of catalyst (k ) and divided by the initial catalyst concentration [Cat]
1
o
as shown in Equation 2.
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k = (k – k )/[Cat]
o
(Equation 2)
2
1
1b
For reactions performed with preꢀincubated AcꢀIHIHIQIꢀNH , peptide was added directly from the
2
pH 2 stock solution to cuvettes containing buffer with ZnCl 24 hours prior to the reaction. The
2
o
o
peptide was incubated at 20 C. The contents of the cuvettes were equilibrated to 20 C for 30
min. prior to catalysis. Reactions were initiated with the addition of substrate dissolved in
acetonitrile (2% acetonitrile in reaction mixture).
For reactions performed with actively assembling AcꢀIHIHIQIꢀNH , reaction cuvettes containing
2
buffer with ZnCl were allowed to equilibrate for 30 min. prior to the reaction. The stock substrate
2
solution and the pH 2 stock solution containing AcꢀIHIHIQIꢀNH were also equilibrated 30 min.
2
before the start of the reaction. Reactions were initiated with the simultaneous addition of
peptide from its pH 2 stock solution and substrate dissolved in acetonitrile.
The concentration of pꢀnitrophenolate (Figures 3, 4 and 6) was calculated from the absorbance
ꢀ1
ꢀ1
measured at 405 nm using the molar extinction coefficient of 12,700 Lmol cm at pH 7.5.
Cryogenic transmission electron microscopy (cryo-TEM). From a stock solution of Acꢀ
IHIHIQIꢀNH2 (0.95 mM) in 10 mM HCl (pH 2), 3.6 ꢁL were withdrawn and simultaneously added
along with 7 ꢁL of stock substrate ZꢀLꢀPheꢀONp solution (0.5 mM) in acetonitrile to 337 ꢁL
Tris/HCl buffer (pH 7.5) containing 2 ꢁL ZnCl (18.3 mM in H O). Immediately after mixing, a
2
droplet (5 ꢂL) of the reaction solution was placed on hydrophilized (plasma treatment using a
BALTEC MED 020 device (Leica Microsystems, Wetzlar, Germany)), perforated carbon filmed
grids (Quantifoil Micro Tools GmbH, Jena, Germany). The excess fluid was blotted off to create
an ultraꢀthin layer (typical thickness of 200ꢀ300 nm) of the solution, which spans the holes of the
support film. The prepared samples were immediately vitrified (25 seconds following sample
preparation) by propelling the grids into liquid ethane at its freezing point (ꢀ184 °C). The vitrified
sample grids were transferred under liquid nitrogen by the use of a Gatan (Pleasanton, CA,
USA) cryoꢀholder (Model 626) into a Tecnai F20 TEM (FEI company, Oregon, USA) equipped
with FEG and operated at 160 kV acceleration voltage. Microscopy was carried out at ꢀ175 °C
sample temperature using the microscope’s low dose protocol at calibrated primary
magnifications of 29k. The defocus was set to be 9.81 ꢁm, respectively. Images were recorded
by the use of a 4kꢀEagle CCD camera (FEI Company, Oregon, USA) at 2k resolution (binning 2).
Acknowledgements
We are grateful for financial support from the Deutsche Forschungsgemeinschaft (KO 1976/5
and SFB 765). Special thanks go to Dr. Allison Berger for proofreading this manuscript.
References
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1]
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24; d) Nanda, V. Nat Chem Biol 2008, 4, 273ꢀ275; e) Kaplan, J.; DeGrado, W. F. Proc Natl Acad Sci
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Biopolymers: Peptide Science
Page 6 of 15
2004, 101, 11566ꢀ11570; f) DeGrado, W. F.; Summa, C. M.; Pavone, V.; Nastri, F.; Lombardi, A.
Annu Rev Biochem 1999, 68, 779ꢀ819.
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a) Cangelosi, V. M.; Deb, A.; PennerꢀHahn, J. E.; Pecoraro, V. L. Angew Chem Int Ed 2014, 53,
7900ꢀ7903; b) Zastrow, M. L.; Pecoraro, V. L. Coord Chem Rev 2013, 257, 2565ꢀ2588; c) Zastrow,
M. L.; Pecoraro, V. L. Nat Chem 2012, 4, 118ꢀ123; d) Tegoni, M.; Yu, F.; Bersellini, M.; Pennerꢀ
Hahn, J. E.; Pecoraro, V. L. Proc Natl Acad Sci 2012, 109, 21234ꢀ21239.
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Korendovych, I. V. Nat Chem 2014, 6, 303ꢀ309; b) Zhang, C.; Xue, X.; Lue Q.; Li, Y.; Yang, K.;
Zhuang, X.; Jiang, Y.; Zhang, J.; Liu, J.; Zuo, J.; Liang, XꢀJ. ACS Nano 2014, 8, 11715ꢀ11723; c)
Baltzer, L.; Broo, K.S.; Nilsson, H.; Nilsson. J Bioorg Med Chem 1999, 7, 83ꢀ91; d) Broo, K. S.;
Nilsson, H.; Nilsson, J.; Flodberg, A.; Baltzer, L.; J Am Chem Soc 1998, 120, 4063ꢀ4068; e) Broo,
K. S.; Brive, L.; Ahlberg, P.; Baltzer. L. J Am Chem Soc 1997, 119, 11362ꢀ11372; f) Fukushima, Y.
Bull Chem Soc Jpn 1996, 69, 431ꢀ439.
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a) Der, D. S.; Edwards, D. R.; Kuhlman, B. Biochemistry 2012, 51, 3933ꢀ3940; b) Nicoll, A. J.;
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Sci 2001, 98, 14274ꢀ14279.
a) Vallee, B. L.; Auld, D. S. Proc Natl Acad Sci 1990, 87, 220ꢀ224; b) Eriksson, A. E.; Jones, T. A.;
Liljas, A. Proteins: Struct, Funct, Genet 1988, 4, 274ꢀ282; c) Woolley, P. Nature 1975, 258, 677ꢀ
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a) Yamaguchi, K.; Kamatari, Y. O.; Fukuoka, M.; Miyaji, R.; Kuwata, K. Biochemistry 2013, 52,
797ꢀ6806; b) Yamaguchi, K.; Matsumoto, T.; Kuwata, K. Biochemistry 2008, 47, 13242ꢀ13251.
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a) Zaramella, D.; Scrimin, P.; Prins, L. J. J Am Chem Soc 2012, 134, 8396ꢀ8399; b) Pedersen, M.
G.; Bersani, A. M. J Math Biol 2010, 60, 267ꢀ283; c) Pengo, P.; Baltzer, L.; Pasquato, L.; Scrimin,
P. Angew Chem 2007, 119, 404ꢀ408; d) Schnell, S.; Mendoza, C. Biophys Chem 2004, 107, 165ꢀ
174; e) Fukushima, Y. Bull Chem Soc Jpn 1996, 69, 2269ꢀ2274; f) Ueoka, R.; Matsumoto, Y.;
Dŏzono, H.; Yano, Y.; Hirasa, H.; Goto, K.; Kato, Y. Tet Lett 1990, 31, 5311ꢀ5314.
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8]
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a) Cleij, M. C.; Drenth, W.; Nolte, R. J. M. J Org Chem 1991, 56, 3883ꢀ3891; b) Ihara, Y.; Okamoto,
M.; Kawamura, Y.; Nakanishi, E.; Nango, M.; Koga, J. J Chem Perkin Trans II 1987, 607ꢀ611; c)
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1983, 1741ꢀ1745; d) Kitano, H.; Sun, ZꢀH.; Ise, N. Macromol 1983, 16, 1823ꢀ1827.
[
[
Hunter, C. A.; Sanders, J. K. M. J Am Chem Soc 1990, 112, 5525ꢀ5534.
10] Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford
University Press, New York, 2000.
[
11] Kuipers, B. J. H.; Gruppen, H. J Agricult Food Chem 2007, 55, 5445ꢀ5451.
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Biopolymers: Peptide Science
Figure 1. Nitrophenyl esters employed as substrates to study the kinetics of catalyzed ester hydrolysis.
Fig. 1
132x117mm (300 x 300 DPI)
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2
Figure 2. CD spectra of AcꢀIHIHIQIꢀNH (10 µM) at pH 2 (10 mM HCl) and at various time points following
o
its addition to 50 mM Tris/HCl pH 7.5 with 100 µM ZnCl
2
at 20 C
Figures 2
91x82mm (150 x 150 DPI)
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Figure 3. pꢀNitrophenolate production upon AcꢀIHIHIQIꢀNH
2
ꢀmediated hydrolysis of pꢀnitrophenyl acetate
) pH 7.5 at
2+
(
pNPA) in the presence of Zno . Reactions were performed in Tris/HCl (50 mM with 100 µM ZnCl
2
20 C with 50 µM substrate and 10 µM AcꢀIHIHIQIꢀNH
2
.
Fig. 3
9
7x83mm (150 x 150 DPI)
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Figure 4. pꢀNitrophenolate production upon AcꢀIHIHIQIꢀNH
2
ꢀmediated hydrolysis of NꢀbenzyloxycarbonylꢀLꢀ
2+
phenylalanine pꢀnitrophenyl ester (ZꢀLꢀPheꢀONp) in the presence of Zn . Reactions were performed in
o
Tris/HCl (50 mM with 100 µM ZnCl
2
) pH 7.5 at 20 C with 10 µM substrate and 10 µM AcꢀIHIHIQIꢀNH
2
.
Fig. 4
1
46x100mm (150 x 150 DPI)
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2
Figure 5. CryoꢀTEM image depicting fibril formation of catalytic peptide AcꢀIHIHIQIꢀNH (10 µM), 25 seconds
after its addition to 50 mM Tris/HCl (pH 7.5) in the presence of ester substrate ZꢀLꢀPheꢀONp (10 µM) and
2+
Zn (100 µM).
Figures 5
7x75mm (150 x 150 DPI)
7
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Figure 6. Schematic representation of AcꢀIHIHIQIꢀNH
2
as it undergoes selfꢀassembly from its initial unfolded
state (A) into a βꢀsheet structures (B), resulting ultimately in a crossꢀβ conformation (C). During this
process, exposed hydrophobic isoleucine residues in A and B are tightly packed together as fibrils are
formed. Zinc atoms, shown as violet spheres, play a crucial role in catalysis, but are not required for Acꢀ
IHIHIQIꢀNH
2
fibril formation.
Fig. 6
199x101mm (150 x 150 DPI)
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Page 13 of 15
Biopolymers: Peptide Science
Figure 7. Production of pꢀnitrophenolate upon the hydrolysis of substrates ZꢀLꢀPheꢀONp and BocꢀLꢀAsnꢀONp
2
+
in the presence of A) AcꢀIHIHIQIꢀNH
2
and Zn in the process of selfꢀassembly and B) AcꢀIHIHIQIꢀNH
2
preꢀ
2+
incubated at pH 7.5 with Zn 24 hours prior to reaction initiation. Reactions were performed in Tris/HCl (50
o
mM with 100 µM ZnCl
2
) pH 7.5 at 20 C with 10 µM initial substrate and 10 µM AcꢀIHIHIQIꢀNH
2
.
Fig. 7
1
70x105mm (150 x 150 DPI)
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Biopolymers: Peptide Science
Page 14 of 15
Table 1. Rate constants corresponding to the hydrolysis of esters pNPA and Lꢀ and Dꢀenantiomers of Zꢀ
PheꢀONp and BocꢀAsnꢀONp in the presence of catalytic peptide AcꢀIHIHIQIꢀNH in its fully fibrillar form
2
2+
o
2+
following a 24 h incubation period with Zn at pH 7.5 and 20 C prior to reaction and with Zn while
actively selfꢀassembling. Reactions were performed at 20 C and pH 7.5 (50 mM Tris/HCl with 100 ꢁM
o
ZnCl ) with 50 ꢁM pNPA and 10 ꢁM ZꢀPheꢀONp and BocꢀAsnꢀONp. A peptide concentration of 10 ꢁM (2
2
peptides form 1 active site = 5 ꢁM catalyst) was used. The apparent k is the observed pseudoꢀfirst order
1
rate constant and does not account for uncatalyzed background hydrolysis. Experiments were performed
in triplicate with the standard deviation shown in parenthesis.
2
+
2+
Uncatalyzed
Ac-IHIHIQI-NH
2
pre-incubated with Zn
Ac-IHIHIQI-NH
Apparent k
2
actively assembling with Zn
-
1
-1
-1 -1
-1
-1 -1
Substrate
k
1
(s )
Apparent k
1
(s )
k
2
(M s )
1
(s )
k
2
(M s )
ꢀ
5
ꢀ4
ꢀ4
pNPA
7 (± 2) x 10
5.1 (± 0.2) x 10
1.11 (± 0.05) x 10
1.00 (± 0.05) x 10
1.21 (± 0.02) x 10
8.52 (± 0.02) x 10
87 ± 4
2.7 (± 0.2) x 10
1.13 (± 0.04) x 10
3.87 (± 0.02) x 10
1.83 (± 0.03) x 10
9.49 (± 0.07) x 10
41 ± 4
ꢀ
ꢀ
ꢀ
ꢀ
3
3
3
3
ꢀ2
ꢀ2
ꢀ2
ꢀ3
3
3
ꢀ1
ꢀ2
ꢀ2
ꢀ3
4
Z-L-Phe-ONp
Z-D-Phe-ONp
Boc-L-Asn-ONp
Boc-D-Asn-ONp
1.53 (± 0.02) x 10
1.53 (± 0.02) x 10
3.80 (± 0.02) x 10
3.80 (± 0.02) x 10
1.9 (± 0.1) x 10
1.7 (± 0.1) x 10
2.23 (± 0.08) x 10
7.43 (± 0.04) x 10
2.90 (± 0.06) x 10
1.14 (± 0.01) x 10
3
3
3
3
2
1.66 (± 0.04) x 10
9.44 (± 0.04) x 10
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Page 15 of 15
Biopolymers: Peptide Science
143x57mm (150 x 150 DPI)
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