Comparing dendritic with linear esterase peptides by screening SPOT arrays for catalysis

Article abstract of DOI:10.1039/c0cc02700f

Fluorescence screening of a 96-membered SPOT library of histidine containing dendritic and linear peptides revealed the remarkable esterolytic activity of short histidine oligomers that show catalytic proficiencies within one order of magnitude of histidine-containing esterase peptide dendrimers. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc02700f

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Comparing dendritic with linear esterase peptides by screening SPOT
arrays for catalysiswz
´
Rasomoy Biswas, Noelie Maillard, Jacob Kofoed and Jean-Louis Reymond*
a
a
b
a
Received 21st July 2010, Accepted 4th October 2010
DOI: 10.1039/c0cc02700f
Fluorescence screening of a 96-membered SPOT library of
histidine containing dendritic and linear peptides revealed the
remarkable esterolytic activity of short histidine oligomers that
show catalytic proficiencies within one order of magnitude of
histidine-containing esterase peptide dendrimers.
Enzyme catalysis arises by cooperativity between amino acid
side chains upon folding of the polypeptide, which enhances
the catalytic power of functional groups and enables substrate
binding by formation of a macromolecular receptor.1 In the
context of enzyme model studies,2 catalytic peptides with
linear,3 cyclic4 or dendritic topologies5 offer a privileged
platform to reconstruct enzyme catalysis in simplified systems.
In our studies of dendritic esterase mimics,6 a combinatorial
approach by split-and-mix synthesis7 led us to histidine
containing peptide dendrimers catalysing the hydrolysis of
fluorogenic esters in aqueous medium with substrate binding
and multiple turnover. In particular catalysts for esterolysis
of 1-acetoxypyrene-3,6,8-trisulfonate
1
exhibit catalytic
proficiencies (kcat/KM) that are 102–105-fold larger than the
2nd order rate constant k2 with the reference small molecule
catalyst 4-methyl imidazole (4MeIm) (Fig. 1).8 These catalysts
include peptide dendrimers using cooperativity between
multiple histidine side chains for binding and catalysis
(e.g. (Ac–HisX)8(DapHisX)4(DapHisX)2DapHisX–NH2, Dap =
L-2,3-diaminopropionic acid branching, A3B: X = b-alanine,
(kcat/KM)/k2 = 28 000 at pH 5.5 with 1, A3C: X = Thr,
(kcat/KM)/k2 = 13 000 at pH 5.5 with 1),9 and peptide
Fig. 1 Ester hydrolysis reaction catalysed by histidine containing
peptides and peptide dendrimers. The imidazole group acts either as a
nucleophile (path a) or general base (path b). See text for amino acid
sequences of individual peptides and peptide dendrimers.
Herein we report the screening of a focused 96-membered
SPOT peptide array library12 on cellulose support leading to
the identification of linear and dendritic peptide catalysts with
remarkable catalytic proficiencies for the hydrolysis of 1.
These experiments show for the first time that screening
SPOT-peptide arrays directly on solid support is an efficient
method to discover peptide catalysts, and reveal the transition
from catalysis by an isolated functional group to cooperative
binding and catalysis in linear peptide and peptide dendrimer
enzyme models.
dendrimers with
a
single catalytic site, e.g. RG3
(Ac–TyrThr)8(DapTrpGly)4(DapArgSer)2DapHisSer–NH2
((kcat/KM)/k2 = 46 at pH 5.5 with 1).10
In RG3 the core catalytic histidine residue is assisted for
substrate binding by a pair of cationic arginines in the first
generation branch and multiple aromatic residues in the outer
dendrimer branches. More recently cyclic peptides with
multiple lysines and a single histidine residue were shown to
catalyse the same reaction thereby acting as ATP and heparin
sensors,4 and octapeptides bearing four histidine residues were
shown to catalyse the hydrolysis of 1.11 We therefore became
interested in directly comparing the esterolytic activity of
histidine containing peptides and peptide dendrimers towards 1.
With the aim of exploring focused sequence variations of
known peptide dendrimer catalysts, we set out to screen a
small library prepared by SPOT synthesis on cellulose using
the fluorogenic hydrolysis of 1 as test reaction. A 96-membered
library was designed featuring variations of esterase dendrimers
A3B, A3C, RG3 and related analogs. The variations included
sequence shuffling as well as repositioning and exchange of
Dap branching against alanine all the way to linear peptides
within a sequence of 11 consecutive couplings. The library
contained linear undecapeptides and 1st, 2nd and 3rd generation
dendrimers with up to 54 amino acids (Tables S1 and S2, ESIz).
SPOT synthesis was performed using automated Fmoc synthesis
a Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012 Berne, Switzerland.
E-mail: jean-louis.reymond@ioc.unibe.ch; Fax: +41 31 631 80 57;
Tel: +41 31 631 43 25
b Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
z Electronic supplementary information (ESI) available: Details of
SPOT library design, synthesis, and screening, synthesis of peptides
and dendrimers and kinetic studies. See DOI: 10.1039/c0cc02700f
c
8746 Chem. Commun., 2010, 46, 8746–8748
This journal is The Royal Society of Chemistry 2010

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on cellulose paper support with a non-cleavable linker in a
customized 96-spot format. All sequences were capped by
acetylation of the N-termini, and the side-chain protecting
groups were removed by global acidic treatment. Following
extensive washing, the peptide SPOTs were punched out and
transferred to a 96-well plate. Activity screening was performed
by immersing the paper disks carrying each library member in a
buffered aqueous solution of substrate 1 (80 mM in 5 mM citrate
pH 5.5) and recording the formation of 1-hydropyrene-3,6,8-
trisulfonate 2 in the solution by fluorescence.
Table 1 Catalytic parameters for selected peptides and dendrimers
c
k
cat/kuncat
(kcat/KM)/k2
/cat
Cpda
NHis
KM/mM
b
/cat
/His
/His
His1
P65d
His2
His3
His4
P60
P60A
His5
His6
P25d
His7
His8
His9
His10
His11
His12
His13
His14
His15
1
1
2
3
4
4
4
5
6
6
7
8
—
120
—
—
620
—
1050
1100
770
—
620
—
3
190
27
130
220
203
203
270
380
2370
790
950
1090
1230
1210
1540
1560
1690
2200
3
190
14
43
55
51
51
54
63
395
115
120
120
125
110
130
120
120
145
280
180
145
155
180
140
47
100
100
90
100
95
110
100
100
86
350
275
190
210
260
250
490
355
375
335
380
320
435
375
370
400
835
1300
1500
2950
2500
3000
3030
3800
3500
5200
4900
5200
6030
Analysis of the screening data (Table S2 and Fig. S1, ESIz)
showed a 45-fold difference in fluorescence intensity reading
between the most and the least active library member. The
least active readings came from dendritic sequences
(Ac–HisX)8(DapGlyGly)4(DapGlyGly)2DapGlyGly– (X = Phe,
Leu, Pro, Thr, Ala, Tyr, Gly, Ser, linked to cellulose), and
sequences containing multiple anionic residues, which inhibit
substrate binding,7e including a simple catalytic triad model
peptide Ac–DSHLDSHLDSH–. The most active reading
came from the histidine undecapeptide His11, followed by
3rd generation dendrimers related to A3B/C carrying multiple
histidines throughout the branches. When considering the
activity per histidine residue, the most active sequences were
(Ac–TyrGly)8(DapTyrSer)4(DapXX)2DapThrHis– for X = Lys
(P65) and X = Arg (P75), which carry only a single catalytic
histidine residue at the core. They were followed by the
histidine undecapeptide His11, Ac–HTHTHTHTHTH– P18,
Ac–HKHKHKHKHKH– P25, Ac–HRHRHRHRHRH– P26,
and Ac–HTAHTAHTAHT– P60.
9
10
11
12
13
14
15
a
All products were prepared by Fmoc-SPPS on TGR resin and
b
purified by preparative RP-HPLC. NHis = number of histidine
residues. k2 is the 2nd order rate constant with 4-methyl imidazole,
k2 = 1.0 Mꢀ1 minꢀ1. The spontaneous background reaction is kuncat
c
=
3.58 ꢁ 10ꢀ5 minꢀ1; /cat = per catalyst; /His = per histidine
residue. Conditions: aq. 5 mM citrate, pH 5.5, 34 1C, 58.5–1000 mM
1. Catalyst concentration: His1–His4: 20 mM; His5–His6, P60, P60A:
15 mM; His7–His14: 5 mM, His15, P25, P65: 3.75 mM. 120 mL assays in
microtiterplate wells were followed by fluorescence (lexc = 450 ꢂ 25 nm,
lem = 530 ꢂ 12 nm). All kinetics were run in triplicate with error
d
ꢂ5–20%, see Table S3 (ESIw) for complete data. P25 and P65
The strong apparent activity of linear peptides in the array
might reflect a higher synthetic efficiency compared to the
dendrimers. Nevertheless, a series of hits and related sequences
were prepared for closer characterization including linear
histidine oligomers up to 15 residues His1–His15, the
cationic His/Lys peptide P25, P60 and its alanine analog
Ac–HAAHAAHAAHA–NH2 (P60A), and peptide dendrimer
P65 which showed the highest apparent activity per histidine
residue. Products were obtained by Fmoc synthesis on
Rink-amide resin and purified by RP-HPLC (Table S3, ESIz).
Catalytic parameters were determined for the hydrolysis of
ester 1 (Table 1, Fig. 2). The activities of purified peptides and
dendrimers in dilute aqueous buffer paralleled the SPOT array
data, suggesting that multivalency on the solid support did not
enhance catalysis during the screening. His1 and His2 behaved
as small molecule catalysts with second order kinetics. All
other peptides starting with His3 obeyed Michaelis–Menten
kinetics with preequilibrium substrate binding and saturation
of catalysis. The strongest catalyst was the pentadecapeptide
His15, with 6000-fold rate acceleration over background
underwent slow acylation at the lysine side-chain with t1/2 E 4–6 h.
Fig. 2 Overview of catalytic proficiencies of linear and dendritic
esterases for the hydrolysis of 1 at pH 5.5. The reactivity increase of
peptide and peptide dendrimer catalysts over 4-methylimidazole per
histidine residue as a function of the number of histidine residues NHis
in the catalysts. Data from this work and ref. 9 and 10.
kcat/kuncat, corresponding to a 2200-fold larger relative catalytic
proficiency (kcat/KM)/k2 compared to 4MeIm. The His–Lys
oligomer P25 also showed a remarkable catalytic proficiency
(kcat/KM)/k2 = 395 due to its stronger substrate binding
(KM = 47 mM, see below). Dendrimer P65 showed the highest
activity per His residue with (kcat/kuncat)/NHis = 620 and
relative catalytic proficiency (kcat/KM)/k2 = 190. However
both P25 and P65 underwent slow acylation at the lysine
side-chains (t1/2 E 4–6 h) and therefore did not behave as true
catalysts.
The oligohistidine peptides showed very good activities but
reached a plateau of relative proficiency per histidine for the
heptamer His7. Further oligomerization did not significantly
increase catalytic proficiency per histidine up to His15, which
remains one order of magnitude less active than peptide
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8746–8748 8747

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contributes to the higher catalytic efficiency per histidine
residue (kcat/kuncat)/NHis observed in the larger histidine
oligomers independent of the increased binding to substrate 1.
In summary, the experiments above represent to our
knowledge the first example of catalyst discovery by screening
a SPOT library on solid support. The library screening showed
that multivalency effects previously observed in peptide
dendrimers for the hydrolysis of pyrenesulfonate ester 1 also
occur in linear peptides. While histidine and its dipeptide
behave as small molecule catalysts, the histidine tripeptide
and longer oligomers show preequilibrium substrate binding
by electrostatic interactions, with catalytic proficiencies
102–103-fold higher than 4-methyl imidazole. Their catalytic
proficiency per histidine residue reaches a plateau at the
level of the heptamer His7, with values within one order of
magnitude of previously identified peptide dendrimers such as
A3B and A3C for the same reaction.
Fig. 3 Inhibition of His15 catalysis of ester 1 hydrolysis by citrate
buffer at pH 5.5. Main plot: Lineweave–Burk plots with increasing
citrate. Inset: Dickson replot of KM/kcat. The 8-fold increase in citrate
from 5 to 40 mM causes a 5-fold increase in KM, a 2-fold increase in
This work was supported financially by the University
of Berne, the Swiss National Science Foundation, and the
Marie-Curie Training Network IBAAC.
k
cat, and a 1.2-fold increase in kuncat. Assay conditions as in Table 1.
Notes and references
dendrimers such as A3B and A3C which also carry 15 histidines.9
Note that the activity per His residue in peptide dendrimer
A3B represents a remarkable multivalency effect which is not
outperformed by dendrimers with higher number of histidines
(Fig. 2).
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Part of the increase in catalytic proficiency in the histidine
linear peptides was caused by stronger binding of substrate 1,
which increased gradually by up to three-fold between the
smallest enzyme model His3 (KM = 280 mM) and the histidine
oligopeptides His7–His15 (KM E 100 mM) or the peptide
dendrimer P65 (KM = 120 mM). The even lower KM of the
polycationic peptide P25 (KM = 42 mM) suggested an electro-
static component in substrate binding, presumably a salt
bridge between cations on the catalyst and the sulfonate
groups of substrate 1, as previously described for multivalent
histidine peptide dendrimer such as A3C.9 Electrostatic
substrate–catalyst interactions were evidenced for His15 by
the observation of a 2-fold increase in KM and 2-fold decrease
in kcat upon addition of 0.5 M KCl to the reaction buffer
(Table S4, ESIz). In addition, catalysis by His15 was inhibited
by citrate (Ki = 13 mM, Fig. 3), and by the tetraanion
pyrene-1,3,6,8-tetrasulfonate (Ki = 280 mM, Fig. S2, ESIz).
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B5.4, which implies that eight of the fifteen histidines are
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oligomerization pKa(His1) E 6.4 > pKa(His2) E 6.1 >
pKa(His3) E 6.0 > pKa(His8) E 5.7 > pKa(His15) E 5.4
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8748 Chem. Commun., 2010, 46, 8746–8748
This journal is The Royal Society of Chemistry 2010