Fluorescent ADP sensing in physiological conditions based on cooperative inhibition of a miniature esterase

Article abstract of DOI:10.1021/ja070734c

This study shows that a cationic esterase mimic, which is responsible for the catalytic hydrolysis of a negatively charged fluorogenic ester, displays different inhibition behaviors in the presence of similar anionic metabolites in water and at physiological pH. On the contrary of the other negatively charged inhibitors tested, ADP strongly slowed down the formation of the fluorescent product by virtue of cooperative inhibition, hence allowing its spectroscopic or visual detection. Our sensor or its derivatives may find future use in biological applications, such as protein kinase activity detection. Copyright

Full text of DOI:10.1021/ja070734c

Published on Web 03/31/2007
Fluorescent ADP Sensing in Physiological Conditions Based on Cooperative
Inhibition of a Miniature Esterase
Laurent Vial and Pascal Dumy*
De´partement de Chimie Mole´culaire, UMR-5250, ICMG FR-2607, CNRS - UniVersite´ Joseph Fourier, BP 53,
38041 Grenoble Cedex 9, France
Received February 1, 2007; E-mail: pascal.dumy@ujf-grenoble.fr
Scheme 1. Miniature Esterase-Catalyzed Ester Hydrolysis8
The development of specific sensors for (poly)anions is of
considerable current interest since they play a fundamental role in
a wide range of biological processes.1 We decided to use enzyme-
based sensors to achieve such a goal.2 If the substrate or the product
of an enzymatic reaction has distinguishable physical properties,
specific inhibition of the catalytic activity by an anion of choice
allows its detection. An interesting variation lies in the use of
enzyme mimic-based sensors since they are a priori less fragile,
more easily available, cheaper, and chemically more flexible (e.g.,
for subsequent immobilization) in comparison with their natural
analogues. The activity and selectivity of native enzymes rely on
complex folds for a precise spatial arrangement of a small number
of catalytically important amino acids. Thus, the presentation of
theses functional groups on small peptides with well-defined
secondary structures is an attractive alternative for the preparation
of effective enzyme mimics.3
We designed the cyclic miniature esterase 1 that displays, in a
controlled geometry4 and on a hypothetical rectangle of ∼5 Å ×
∼7 Å, three distinct positive charges (via the presence of lysines)
for molecular recognition and a nucleophilic/basic center (via the
presence of a histidine residue) for catalytic hydrolysis of the planar
compound 2, which displays itself three anionic charges and an
ester function on a hypothetical rectangle of similar size (Scheme
between MeIm and 1, this rate enhancement was attributed to either
1).5 We reasoned that this good topology match occurring between
nucleophilic or general base catalysis assisted by molecular
opposite charges of both partners should lead to the close proximity
recognition of the substrate; its modest magnitude probably also
of the nucleophilic/basic nitrogen and the ester function and, as a
indicating that no cooperative effect occurs between amino acid
result, should be responsible for ester hydrolysis rate enhancement.
In addition, the hydrolysis product 3 (cascade blue) is an excellent
residues.3c
Then, we chose and tested a series of various metabolites carrying
fluorescer, furnishing therefore an optical signal that could poten-
several negative charges (from two to six) as potential inhibitors
tially be modulated in presence of anionic species acting as
of miniature esterase 1. Whereas orthophosphate (Pi), AMP, D-myo-
inhibitors.6
inositol-1,4,5-triphosphate (IP3), citric acid, and ATP turned out to
be null to moderate inhibitors (inhibition constants KI up to 244
µM), ADP exhibited an unexpected parabolic Dixon plot (Figure
1C). This curvature is indicative of cooperative inhibition when
Synthesis of peptide 1 was carried out by Fmoc strategy on
chlorotrityl resin (500 mg, loading: 0.42 mmol/g), affording the
protected linear peptide H-K(boc)-A-H(tr)-P-G-K(boc)-A-K(boc)-
P-G-OH. Cyclization under high dilution conditions (0.5 mM) and
more than one inhibitor molecule binds simultaneously to a
subsequent deprotection gave the final product in good quantity
catalyst.9,10 Hill plot analysis of the data (Figure 1D) was conducted
and suggested a 2:1 complex between ADP and peptide 1 (Hill
coefficient nH ) 2.04 and global dissociation constant KD ) 52
µM).11 A preliminary modeling study did not give us insight into
the structure of such a ternary complex, which is probably the result
of the subtle interplay of noncovalent interactions (e.g., stacking
interactions between the nucleobases).
(143 mg, overall yield 66%).7
Hydrolysis of 2 in water (Tris buffer, pH 7.4) was followed by
fluorescence in a 96-well microliter-plate setup (λexc ) 460 nm,
λem ) 530 nm). Enzyme-like kinetics was observed in the presence
of 1 with multiple turnover activity (Figure 1A). A Michaelis-
Menten model was applied to determine substrate binding KM
)
135 µM, pseudo-first-order rate constant kcat ) 0.0498 min-1, rate
enhancement kcat/kuncat ) 520, and transition state dissociation
constant KTS ) KM/(kcat/kuncat) ) 0.260 µM (Figure 1B). In the same
conditions, catalysis by 4(5)-methylimidazole (MeIm) as reference
This large difference in affinity between 1 and the different
anionic inhibitors, associated with the colorful property of 3,
permitted a convenient “naked-eye” detection of ADP directly from
kinetic assays. In conditions where inhibitors are in enough excess
relatively to 1 and 2 (g800 µM for 200 µM of substrate 2 and 5
µM of peptide 1), Pi, AMP, IP3, ATP, or citric acid all lead to
yellow-colored wells as a result of the formation of 3, while the
catalyst was very inefficient (Figure 1A). A second-order kMeIm
)
0.0124 mM-1 min-1 was measured at millimolar MeIm concentra-
tions and used to calculate the catalytic enhancement (kcat/KM)/kMeIm
) 30. On the basis of a conservative mechanistic hypothesis
9
4884
J. AM. CHEM. SOC. 2007, 129, 4884-4885
10.1021/ja070734c CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
find future use in biological applications, such as protein kinase
activity detection.14
Acknowledgment. This work was supported by the Centre
National de la Recherche Scientifique, the Universite´ Joseph Fourier
de Grenoble, and the Institut Universitaire de France.
Supporting Information Available: Detailed synthetic and kinetic
assays procedures, ES-MS spectrum of compound 1 (PDF). This mater-
ial is available free of charge via the Internet at http://pubs.acs.org.
References
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Figure 1. (A) Time plot formation of 3 from the hydrolysis of ester 2
(200 µM) in the presence of catalytic peptide 1 (red) or 4-methylimidazole
(blue), and background reaction (green). (B) Lineweaver-Burk plot of the
hydrolysis of ester 2 with catalytic peptide 1. (C) Dixon plots of the activity
inhibition of peptide 1 by Pi (dark green), citric acid (light green), IP3 (dark
blue), AMP (light blue), ATP (yellow), and ADP (red). (D) Hill plot of the
activity inhibition of catalytic peptide 1 by ADP. General conditions: 5
µM catalyst 1 or 4(5)-methylimidazole, 24 °C, 20 mM Tris buffer pH 7.4.
(4) The presence of two prolylglycine sequences as â type II turn inducers
constrains the peptide conformation into an antiparallel â-sheet. For a
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(10) Peptide 1 did not catalyze the hydrolysis of ADP in the conditions used
in this study.
Figure 2. (A) Fluorescence versus time plot (λexc ) 460 nm, λem ) 530
nm) showing the formation of 3 from the hydrolysis of ester 2 in the
presence of catalytic peptide 1 and ADP (red), ATP (yellow), AMP (light
blue), citric acid (light green), IP3 (dark blue), or Pi (dark green). RFU )
relative fluorescence unit.12 (B) Kinetic assay wells. From top to bottom:
ATP, ADP, AMP, Pi, IP3, and citric acid. General conditions: 800 µM
inhibitor, 5 µM peptide 1, and 200 µM ester 2 in 20 mM Tris buffer pH
7.4, 24 °C.
(11) Parameters were calculated using the Hill equation: log [V/(Vm - V] )
-nH log [inhibitor] + log KD, where nH, Vm, and KD are the Hill coefficient,
the maximum velocity, and the global dissociation constant, respectively.
(12) The curve shift on the y-axis at t ) 0 is related to the time delay that
occurred between the sample preparation and the beginning of the
spectroscopic measurement.
ADP containing solution remains completely uncolored, even after
24 h reaction (Figure 2A,B).
This study shows that a cationic esterase mimic (1), which is
responsible for the catalytic hydrolysis of a negatively charged
fluorogenic ester (2), displays different inhibition behaviors in the
presence of similar anionic metabolites in water and at physiological
pH. On the contrary of the other negatively charged inhibitors tested,
ADP strongly slowed down the formation of the fluorescent product
(3) by virtue of cooperative inhibition, hence allowing its spectro-
scopic or visual detection. This result is remarkable since such ADP
sensors, with good selectivity against ATP in particular, are poorly
represented in the literature.13 Our sensor or its derivatives may
(13) For nice examples, see: (a) Aguilar, J. A.; Garcia-Espana, E.; Guerrero,
J. A.; Luis, S. V.; Llinares, J. M.; Miravet, J. F.; Ramirez, J. A.; Soriano,
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Goddard, J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22, 363-370.
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