Profiling primary protease specificity by peptide synthesis on a solid support

Article abstract of DOI:10.1002/anie.200353367

Reverse screening: A greatly simplified primary screening of protease specificity has been achieved by monitoring the fluorescence during the protease-catalyzed coupling of amino acids instead of peptide hydrolysis on a solid support (see picture, AA = amino acid). This approach paves the way for flexible, rapid, high-throughput identification and characterization of proteases without the need for expensively labeled peptide arrays.

Full text of DOI:10.1002/anie.200353367

Communications
Enzyme Screening
Profiling Primary Protease Specificity by Peptide
Synthesis on a Solid Support**
Ron H. P. DoezØ, Beatrice A. Maltman, Claire L. Egan,
Rein V. Ulijn, and Sabine L. Flitsch*
An estimated 2–3% of the mammalian proteome consists of
proteases: enzymes that hydrolyze proteins and peptides.[1]
Many of these enzymes are involved in essential physiological
functions such as immunological defense and cell differ-
entiation. Several proteases are involved in disease states,
such as HIV, Alzheimerꢀs disease, hepatitis C, Candida
infections, and pancreatitis.[2] In addition, proteases have
found applications as (industrial) biocatalysts in synthetic
organic chemistry as a result of their ability to selectively
hydrolyze (or synthesize) peptide bonds with high specificity
[*] R. H. P. DoezØ, Dr. B. A. Maltman, C. L. Egan, Prof. S. L. Flitsch
School of Chemistry
The University of Edinburgh
King’s Buildings, West Mains Road, Edinburgh, EH9 3JJ (UK)
Fax: (+44)131-650-4737
E-mail: slf01@staffmail.ed.ac.uk
Dr. R. V. Ulijn
Manchester Materials Science Centre
University of Manchester/UMIST
Grosvenor Street, Manchester, M1 7HS (UK)
[**] This work was sponsored by the Biotechnology and Biological
Sciences Research Council, the European Union, and the Wellcome
Trust. We would also like to thank Polymer Laboratories (UK) for the
supply of PEGA1900 and Dr. Ayhan Celik for help with the
fluorescence measurements.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200353367
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under mild conditions.[3] Given the large number of proteases
and their importance in both synthetic chemistry and
biomedicinal science, high-throughput screening methods
for the measurement of protease specificity are required.
Key to the characterization of this class of enzymes is the
definition of substrate specificity, that is, the selectivity for
amino acids flanking the scissile peptide bond. The number of
potential substrates for proteases can be large. There are 400
possible dipeptide sequences if only the “primary” amino
acids directly adjacent to the scissile bond are considered.
This number increases to 160000 when secondary sites are
considered. Several methods have been developed to deter-
mine the specificity of proteases. Large peptide libraries are
used where the library members are attached on solid
supports such as polymer beads[4] or microarrays.[5] For
example, Meldal and co-workers have developed screening
methods for large combinatorial peptide (or peptide mimic)
libraries by using quenched fluorogenic labels
ports. PEGA1900 has been identified as a suitable biologically
inert material where all reactive sites on the polymer are
accessible to small enzymes. Literature reports have shown
that proteases are catalytically active within PEGA1900
beads.[4,6]
To assess the validity of our approach, the selectivity of
the protease thermolysin from Bacillus thermoproteolyticus
Rokko in the hydrolysis and synthesis directions was com-
pared by analytical HPLC analysis. Attachment of two amino
acids (Asp and Phe) and two Fmoc-protected dipeptides
(Fmoc–Asp–Phe and Fmoc–Phe–Asp) through the Wang
linker to PEGA1900 resin provided solid-supported substrates
for the synthesis and hydrolysis reactions, respectively.
Analysis of the products (Fmoc–Phe–Asp and Fmoc–Asp–
Phe after acidic cleavage of the Wang linker or of Fmoc–Asp
and Fmoc–Phe, respectively) was performed by LC-MS.
Figure 2 shows that thermolysin-catalyzed synthesis and
that become fluorescent upon peptide hydrol-
ysis. Both the synthesis and the biological
screening of these libraries could be carried
out directly on polyacrylamide/poly(ethylene
glycol) (PEGA copolymer) polymers that are
compatible with enzyme activity.[4] Recently, the
research groups of Ellman and Yao independ-
ently developed successful peptide chips where
the members of a peptide library were directly
arrayed onto glass slides. This method is so far
restricted to the analysis of the specificity for
amino acids positioned on the carboxylic side of
the cleaved peptide bond.[5a,b]
Here we describe a simple new method for
the profiling of the primary specificities of
Figure 2. Thermolysin-catalyzed hydrolysis and synthesis of Fmoc–Asp–Phe–Wang–PEGA1900 and
Fmoc–Phe–Asp–Wang–PEGA1900. a) Hydrolysis of Fmoc–Asp–Phe (circles) is favored over hydrol-
ysis of Fmoc–Phe–Asp (squares). b) In the presence of excess Fmoc–Asp or Fmoc–Phe, synthesis
of Fmoc–Asp–Phe (circles) is favored over synthesis of Fmoc–Phe–Asp (squares). Analyses were
performed by LC-MS.
proteases. The primary specificity describes the protease
preference for the amino acids on either side of the amide
bond (P1 and P01).[6] The assay is based on our recently
reported discovery that the peptide hydrolysis equilibrium
can be reversed toward peptide synthesis on PEGA polymer
beads.[7] Instead of studying the cleavage of peptides, the
reverse reaction, that is, the protease-catalyzed coupling of
amino acids is monitored (Figure 1).
hydrolysis follow the same selectivity, with a marked prefer-
ence for the Fmoc–Asp–Phe over the Fmoc–Phe–Asp
sequence. The rates of the reactions appear similar because
the kinetics are dominated by the rate-limiting diffusion of
enzyme into the beads, as shown previously.[8]
For comparison of two different proteases, commercially
available bovine pancreatic a-chymotrypsin and thermolysin
from B. thermoproteolyticus Rokko, which display opposite
but complementary specificities for certain P1/P01 amino acid
combinations were selected. While a-chymotrypsin is selec-
tive for aromatic amino acids in the P1 position, it is rather
unspecific for the P01 residue. By contrast, thermolysin exhibits
a preference for large hydrophobic residues in the P01 position
and is nonspecific for the P1 residue. Hence, one would expect
hydrolysis/synthesis of the amide bond in Asp–Phe to be
catalyzed preferentially by thermolysin and that of Phe–Asp
by a-chymotrypsin.[9]
Biocompatible polymer materials are required for the
successful analysis of enzymatic reactions on polymer sup-
To test whether this specificity could be measured with the
present method of peptide synthesis, PEGA-immobilized Phe
and Asp were exposed to Fmoc–Asp and Fmoc–Phe in the
presence of either of the two enzymes. After overnight
incubation, the relative fluorescence intensities were meas-
ured; these directly indicated the level of protease-catalyzed
peptide synthesis. Figure 3 shows that the relative fluores-
cence intensities observed indeed corresponded to the
reported specificities for both enzymes. These experiments
Figure 1. Protease specificity assay on a microtiter plate. Each well
contains 1 of the 20 different P10 amino acids (AA) directly linked to
PEGA-NH2 through the carboxylic acid terminus. The P1 amino acid
AA’ carries a fluorescent 9-fluorenylmethoxycarbonyl (Fmoc) protecting
group and is added in a buffer solution containing the protease of
interest. After incubation of the beads for 16 h at RT, they are washed
and the Fmoc fluorescence is read by using a plate reader.
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Communications
Figure 3. Comparison of substrate selectivity of two complementary
proteases. Reactions were performed as outlined in Figure 1 with
either Asp or Phe in positions P1 or P01. “Relative fluorescence” is a
measure of transfer of the respective P1 amino acid by thermolysin
(gray) or chymotrypsin (black). The experiment shows that thermolysin
favors formation of Fmoc–Asp–Phe–PEGA while chymotrypsin favors
formation of Fmoc–Phe–Asp–PEGA.
suggest that solid-phase screening through peptide synthesis
can give the same specificity as observed in peptide hydrol-
ysis. Indeed, protease specificity is expected to be identical in
either the hydrolysis or synthesis directions because the same
transition states are involved.
The assay was then applied in a 96-well microtiter plate
format by using thermolysin as a model system. Thermolysin
specificity against three P1 amino acids carrying side chains
with different physical properties was monitored: phenyl-
alanine (aromatic side chain), aspartic acid (negatively
charged), and glycine (smallest amino acid). The fluorescence
intensities were measured, compared directly (Figure 4, top
images), normalized, and converted into a gray-scale (bottom
image) with white representing the highest fluorescence
intensity and black representing no signal above the back-
ground level.
The reported specificity for large hydrophobic amino
acids, specifically leucine (L), isoleucine (I), methionine (M),
and phenylalanine (F) is indeed observed for the three P1
amino acids studied here. Alanine (A), glycine (G), and
tyrosine (Y) were recognized to a lesser extent as they are
smaller or less hydrophobic than the preferred substrates.
Tryptophan (W) appears to be too bulky to fit into the active
site and is not accepted by thermolysin.[10]
Figure 4 also shows that the nature of P1 significantly
affects the selectivity for P10 in thermolysin. It appears that a
large hydrophobic phenyl group in the P1 position restricts P10
to large hydrophobic amino acids (mainly Ile, Leu, and to a
lesser extent Val, Met, and Phe), while for smaller Gly
residues the substrate specificity is more relaxed and a wider
range of substrates are accepted. This highlights the impor-
tance of screening all combinations of P1 and P01, rather than
both sites independently, to fully characterize substrate
specificity.
In summary, we have presented a new approach to
screening for protease specificity. Primary screening of
protease specificity is greatly simplified by taking advantage
of the reversal of peptide hydrolysis (that is, peptide syn-
thesis) on a solid support. The choice of label for each added
amino acid is flexible; indeed, different fluorescent labels
could be used for different amino acids and thus might aid
simultaneous detection and analysis. The methodology has
been demonstrated for a microtiter plate system but should be
also applicable to the array format. Specificity for sites away
from the primary positions can also be monitored by
Figure 4. Primary thermolysin specificity as determined by reverse pep-
tide hydrolysis on a solid support. Three different P1 amino acids were
supplied as Fmocderivatives in separate experiments and all of the
natural P01 amino acids were directly linked to PEGA1900. The histo-
grams show the average fluorescence response obtained over three
measurements. (The large error bars on the data are mainly as a result
of inaccuracies resulting from dispensing bead suspensions in micro-
titer plates.) The bottom image shows the relative fluorescence nor-
malized against the P01 amino acid which gave rise to the highest inten-
sity. Lighter shades indicate increased fluorescence observed.
incubation with di-, tri-, and polypeptides instead of Fmoc-
protected amino acids. Work on such an extension of the
methodology is currently in progress. We believe that the
present approach paves the way for flexible rapid high-
throughput identification and characterization of proteases
without the need for expensively labeled peptide arrays.
Received: November 20, 2003
Revised: March 23, 2004 [Z53367]
Keywords: enzymes · fluorescent probes · high-throughput
.
screening · hydrolysis · substrate specificity
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