Structural and Functional Analysis of the Complex between Citrate and the Zinc Peptidase Carboxypeptidase A.
ABSTRACT A high-resolution carboxypeptidase-Zn(2+)-citrate complex was studied by X-ray diffraction and enzyme kinetics for the first time. The citrate molecule acts as a competitive inhibitor of this benchmark zinc-dependent peptidase, chelating the catalytic zinc ion in the active site of the enzyme and inducing a conformational change such that carboxypeptidase adopts the conformation expected to occur by substrate binding. Citrate adopts an extended conformation with half of the molecule facing the zinc ion, while the other half is docked in the S1' hydrophobic specificity pocket of the enzyme, in contrast with the binding mode expected for a substrate like phenylalanine or a peptidomimetic inhibitor like benzylsuccinic acid. Combined structural and enzymatic analysis describes the characteristics of the binding of this ligand that, acting against physiologically relevant zinc-dependent proteases, may serve as a general model in the design of new drug-protecting molecules for the oral delivery of drugs of peptide origin.
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ABSTRACT: The first metallocarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metallocarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive carboxypeptidases CPA and CPB.Journal of Biological Chemistry 09/2011; 286(45):39023-32. · 4.65 Impact Factor
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ABSTRACT: The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.Journal of Biological Chemistry 12/2011; 287(9):6503-17. · 4.65 Impact Factor
SAGE-Hindawi Access to Research
Volume 2011, Article ID 128676, 8 pages
Structuraland FunctionalAnalysisof theComplex between
Citrate andthe ZincPeptidase CarboxypeptidaseA
DanielFern´ andez,1,2EsterBoix,1IrantzuPallar` es,1,2
Francesc X. Avil´ es,1,2and Josep Vendrell1,2
1Departament de Bioqu´ ımica i Biologia Molecular, Facultat de Bioci` encies, Universitat Aut` onoma de Barcelona,
08193 Bellaterra, Spain
2Institut de Biotecnologia i de Biomedicina, Universitat Aut` onoma de Barcelona, 08193 Bellaterra, Spain
Correspondence should be addressed to Ester Boix, email@example.com and Josep Vendrell, firstname.lastname@example.org
Received 5 April 2011; Accepted 30 May 2011
Academic Editor: John J. Tanner
Copyright © 2011 Daniel Fern´ andez et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
A high-resolution carboxypeptidase-Zn2+-citrate complex was studied by X-ray diffraction and enzyme kinetics for the first time.
The citrate molecule acts as a competitive inhibitor of this benchmark zinc-dependent peptidase, chelating the catalytic zinc ion in
the active site of the enzyme and inducing a conformational change such that carboxypeptidase adopts the conformation expected
to occur by substrate binding. Citrate adopts an extended conformation with half of the molecule facing the zinc ion, while the
other half is docked in the S1?hydrophobic specificity pocket of the enzyme, in contrast with the binding mode expected for a
substrate like phenylalanine or a peptidomimetic inhibitor like benzylsuccinic acid. Combined structural and enzymatic analysis
describes the characteristics of the binding of this ligand that, acting against physiologically relevant zinc-dependent proteases,
may serve as a general model in the design of new drug-protecting molecules for the oral delivery of drugs of peptide origin.
Carboxypeptidase A (CPA) is the canonical form of the M14
family of peptidases, an important group of physiologically
relevant enzymes . M14 peptidases such as CPA and
CPB, synthesized in the pancreas and secreted into the
gastrointestinal tract, are the major zinc-dependent enzymes
responsible for the release of the C-terminal amino acid
residue from food protein . Carboxypeptidase activity in
the gastrointestinal tract may be modulated by dietary zinc
[3, 4] or can be inhibited by some organic food components
[5–7]. Also, changes in the genomic expression levels may
control carboxypeptidase enzyme activity in digestion . In
other compartments of the body, different CPs participate
in tightly regulated processes such as, for instance, the
maturation of physiologically active peptide hormones.
Moreover, M14 peptidases are therapeutically interesting
molecules because of their involvement in acute pancreatitis
[9–11], cancer [12–14], diabetes [15, 16], fibrinolysis ,
and inflammation .
Carboxypeptidase A structure reveals that the catalytic
zinc ion is coordinated by three protein residues and by one
water molecule which is displaced upon substrate binding.
The substrate is held in the binding pocket through an
ion pair between its C-terminal carboxylate group and
the guanidinium side chain of Arg145 and two additional
hydrogen bonds with Asp144 and Tyr248. The enzyme-
substrate interaction is additionally stabilized by the overall
hydrophobic character of the binding pocket which reduces
competitive solvation by water molecules . The CPA
catalytic mechanism has attracted much attention as a
model for zinc-dependent proteases and as such routinely
investigated [20–25]. In the two most widely accepted
role either by activating the amide carbonyl of the substrate
or by creating a zinc-water nucleophile that directly attacks
the amide group . Chelation of the zinc ion is an
important aspect in the development of chemotherapeutic
agents targeted to this class of enzymes [27, 28] as well as
in the development of new drug protective molecules for the
administration of pharmacologically important peptide and
protein drugs [29, 30].
In the course of our investigations on M14 peptidases
and organic synthetic inhibitors , we obtained a high
an interaction can be understood from the metal-chelating
properties of the citrate anion as citrate, a major component
in snake venom, is believed to inactivate metal proteases in
the prey assisting in venom toxin movement . Database
searches revealed only a few instances in which a biological
macromolecule-Zn2+-citrate system had been studied at
the structural level; surprisingly, none of these involve a
biologically active zinc-dependent peptidase. It is interesting
to note that in a recent investigation citrate inhibition of
carboxypeptidase enzymatic activity has been linked to drug
permeation-enhancing properties of the carrier molecule
. Thus, the high-resolution structural determination of
the CPA-Zn2+-citrate ternary complex presented here along
with the kinetic evaluation of citrate inhibition of the CPA
catalytic mechanism is intended to gain further insights into
the interactions between this coligand of pharmaceutical use
and the model zinc-dependent intestinal protease.
2.1. Protein Crystallization and Structure Solution. Lyophi-
lized bovine pancreas carboxypeptidase A (Sigma, St. Louis,
MO) was dissolved in buffer 0.02M Tris pH 7.5/1.2M LiCl
and then desalted and concentrated in Centricon centrifugal
filter devices (Millipore, Billerica, MA, USA) against 5mM
Tris, pH 7.5. During a screening to analyze the structures of
CPA-organic inhibitor complexes, CPA crystals were grown
from a 2μL:2μL mixture of enzyme solution (10mg/mL
in 0.02M Tris, pH 7.5) and precipitant (20% PEG 3, 350,
0.2M NH4Cl, 0.02M Tris, pH 7.0) by the vapour diffusion
method. Crystals suitable for X-ray diffraction analysis were
grownin thesitting drop setting in anincubatoratthesteady
temperature of 289K. The largest crystals were harvested
and transferred to a new 2μL drop containing the reservoir
solution plus a millimolar amount of inhibitor solution.
As crystal damage was evident, sequential soakings with
were harvested, briefly bathed in cryoprotectant buffer (i.e.,
the reservoir solution with added 30% glycerol), flash frozen
in the nitrogen stream, and diffracted. Structure determi-
nation was made with the molecular replacement method
using native CPA (PDB entry 2ctb) as the model. Extra
electron density accounting for three planar carboxylate
groups was clearly evident in the region near the catalytic
zinc ion. The shape of this density was easily interpreted
as a citrate molecule. Sodium citrate, a buffer used to
dissolve the inhibitor, would be the probable source of the
ligand seen in the electron density maps. No trace of other
inhibitor molecules is seen in the maps. The refinement
progressed to convergence and reached a good agreement
between the model and the experimental data as shown
by the statistics listed in Table 1. Protein X-ray diffraction
experiments were performed at the X11 and X13 beamlines
of the EMBL (European Molecular Biology Laboratory)-
Outstation at DESY (Deutsches Elektronen Synchrotron),
Hamburg, Germany. X-ray diffraction data was processed
The CCP4 suite  and programs PHASER  and
REFMAC  were used in different stages of structure
solution and refinement. MolProbity  and PROCHECK
 were employed to check the protein coordinates against
reference geometric values. Model building and map fitting
weremade withCoot .Allthefinalfigureswereprepared
with Pymol (http://www.pymol.org/).
2.2. Kinetic Analysis. Benzylsuccinic acid (BzlSA), sodium
citrate, and other reagents were from Sigma (St. Louis, MO).
AAFP substrate was from Bachem (Bubendorf, Switzerland).
The working solutions included various concentrations of
each compound and one AAFP substrate concentration,
usually at the Km value. The final assayed concentrations
were stepwise varied between 1nM and 10mM (for BzlSA)
and between 10μM and 100mM (for citrate). One blank
well was used as the control without inhibitor, and the
experiments were performed at least by duplicate. The
enzyme concentration was adjusted for the kinetic mea-
surements to take place within the initial velocity regime.
Changes in absorbance due to proteolytic activity were
followed continuously in a multimode plate reader Wallac
1420 Victor3 system (PerkinElmer Life Sciences, Waltham,
MA, USA). The kinetic measurements were carried out at
Pad Version 5.0 package (GraphPad Software, San Diego,
CA, USA). After ensuring a good agreement between the
experimental observations and the inhibition model, the Ki
value was calculated from the IC50 following the procedure
of Cheng and Prussoff . The goodness of fit between
was excellent as indicated by the R2parameters, 0.9971 and
0.9822 for BzlSA and citrate, respectively, and by the span
of the 95% confidence interval, in which the Ki values lie
3.Results and Discussion
3.1. Analysis of Structural Data. Although citrate anion is
widely used as a buffer in crystal structure determinations,
out of the few examples of zinc-dependent proteases bound
to this ligand that can be found in the RCSB PDB database,
only two hits, namely, human procarboxypeptidase B (entry
1kwm) and porcine procarboxypeptidase A (1pca), were
retrieved when searching for carboxypeptidases. Neither
of these structures shows citrate in coordination with the
catalytic zinc ion, presumably because the presence of the
prodomain in both zymogen structures hinders the access to
the active site of the enzyme, but the citrate anion resides
on the protein’s surface exposed to external solvent water
molecules. This observation suggests that citrate does bind
to exosites in digestive immature proteases, but no structural
evidence on its ability to bind to the catalytic machinery
of a mature enzyme is available yet. A further query to
Table 1: Statistics of data collection and refinement for citrate-bound CPA.
Wavelength used during data collection
Unit cell constants
a = 40.61˚ A, b = 57.01˚ A,
c = 60.61˚ A
α = 90.0◦, β = 102.4◦, γ = 90.0◦
Number of measured reflections
Number of unique reflections
Completeness & multiplicity (overall/outermost shell)
I/σI (overall/outermost shell)
Reflections used for refinement (total/test set)
Deviation from ideality
r.m.s.d. bond lengths
r.m.s.d. bond angles
Number of protein atoms/total atoms
B-factor statistics (˚ A2)
Overall B-factor/Wilson plot B-factor
Catalytic domain, main/side chain
Zn2+(1 in total/1mol per monomer)
Citrate atoms (13 in total/1mol per monomer)
Glycerol atoms (36 in total/6mols per monomer)
Solvent atoms (214 in total)
Residues with bad bonds/angles
Notes:aRmerge= ΣhklΣj=1to N |Ihkl−Ihkl, (j)|/ΣhklΣj=1to NIhkl(j), where N is the redundancy of the data. The outermost shell is 1.75–1.70˚ A.
bRfactor= Σhkl||Fobs| − |Fcalc||/Σhkl|Fobs|, where the Fobsand Fcalcare the observed and calculated structure factor amplitudes of reflection hkl.
cRfree= is equal to Rfactorfor a randomly selected 3.3% subset of reflections that were not used in refinement.
dAccording to Molprobity .
96.7% (294 of 304 residues)
99.7% (303 of 304 residues)
0.3% (1 of 304 residues, Ser-199)
the RSCB PDB revealed a total of 13 macromolecule-zinc-
citrate structures, none of which involve digestive enzymes.
Therefore, the present crystal structure determination rep-
resents the first example of a metal-dependent, fully active,
physiologically relevant protease in a ternary complex with
citrate. A crystal belonging to the monoclinic space group
P21that diffracted beyond 1.70˚ A was characterized in this
study; crystallographic and refinement statistics details are
presented in Table 1. The atomic coordinates and structure
factors have been validated and deposited with the RCSB
PDB with accession code 3kgq.
3.2. The Binding of Citrate to the Catalytic Centre in the
CPA Active Site. One citrate anion per carboxypeptidase-
Zn2+cation was identified from the electron density maps
(Figure 1). Citrate binds in an extended conformation to
the catalytic centre of the enzyme’s active site, where the
amide bond is be hydrolyzed, across subsites S1 (defined
by residues Arg127 and Glu270, which are involved in the
nucleophilic attack of the scissile amide carbon) and S1?
(Asn144, Arg145, and Tyr248, which anchor and stabilize the
peptide substrate). All but one of the four functional groups
from citrate make important interactions with the metal and
several ligands from the protein (Table 2 and Figure 2).
The zinc ion is bidentately chelated by citrate oxygen
atoms O1 and O2, both atoms lying in the proximity
of two zinc ligands from the protein (His69 and Glu72).
Oxygen atoms from two different carboxylate groups are
hydrogen bonded to Arg127. The nucleophile Glu270 forms
a strong hydrogen bond to one carboxylate and the hydroxyl
group. An ion pair is formed between oxygen atoms O5
and O6 from citrate and the guanidinium side chain of
Figure 1: Partial view of the carboxypeptidase-Zn2+-citrate com-
plex structure localized around the citrate ligand. The ligand is
coloured green while protein atoms are in grey. Oxygen and
nitrogen atoms are red and blue, respectively. The Zn2+ion is
depicted as a sphere in yellow. Relevant residues for binding are
shown as sticks and labelled. Polar interactions are shown as solid
lines in black. The 2Fo-Fcmap, calculated by deleting citrate and
Zn2+coordinates, is contoured at 1.0 sigma (gold mesh).
Table 2: Geometric details of the interaction between protein
residues and the citrate ligand.
Protein residue ID
Distance (˚ A)
Arg145. These atoms make additional interactions with the
other S1?residues, Asn144 and Tyr248. The latter residue
has been identified as a key player in the CPA catalyzed
mechanism by site-specific mutagenesis studies . The
conformational change suffered by Tyr248 upon ligand
binding is well documented: the aromatic ring switches by
120◦its location between the solvent exposed “open” to the
Figure 2: The structures of CPA bound to the peptidomimetic
BzlSA (a) and citrate (b) are schematically shown. Important
residues for binding the zinc ion (sphere) are included. Water
molecules shielding citrate are shown as wavy lines.
“closed” conformation with the phenolic hydroxyl pointing
to the catalytic zinc [41, 42]. In this paper, Tyr248 is in the
closed conformation expected to occur when the active site
of the enzyme is occupied. As seen in other CPA-bound
structures, the water molecule is displaced from the zinc
coordination sphere by a carboxylate from the ligand and
the citrate molecule binds to CPA in a manner reminiscent
of the succinate-based ligands such as the benchmark
inhibitor benzylsuccinic acid (BzlSA). As will be commented
next, while the aromatic ring of BzlSA is buried in the
hydrophobic pocket of CPA, the carboxylate group of citrate
that occupies the corresponding edge of the molecule adapts
its conformation to this specific enzyme’s environment.
3.3. Citrate Burial in the Hydrophobic Pocket of CPA. Like
citrate, BzlSA chelates zinc and forms an ion pair with
Arg145 and hydrogen bonds to catalytically key residues
like Tyr248, but the two molecules also display considerable
differences in binding to the enzyme (Figure 3). From
Figure 3: Comparison of carboxypeptidase-bound structures. (a)
CPA in complex with BzlSA (PDB entry 1cbx). (b) CPA with bound
citrate (this work). BzlSA and citrate are shown magenta and green,
respectively. Ala250 and Ile255, enclosing the ligand, are in orange.
Note the location of two water molecules (spheres in red) at (b),
masking hydrophobic residues at the bottom of the active site.
Hydrogen bond interactions are as dashed lines in black. Both
structures are shown in similar orientation.
inspection of the crystal structure in complex to CPA (PDB
entry 1cbx), BzlSA extends towards the bottom of the active
site cleft with an aromatic ring that mimics a phenylalanine
residue. Side chains Leu203, Ile243, Ala250, Gly253, Ile255,
and Thr268 define the hydrophobic pocket that harbours the
specificity-determinant residue in the M14 peptidase family,
which for CPA is Ile255. This hydrophobic pocket is best
suited to accommodate an aromatic ring, and Ile255 makes
hydrophobic interactions with this part of the inhibitor
The third carboxylate group from citrate is the only one
shielded from contact to CPA. Each one of the oxygen atoms
of the carboxylate group, O3 and O4, makes strong hydrogen
bonds to water molecules, which in turn are hydrogen
bonded to otherwatermoleculesand amino acidside chains.
Thus, this ionisable group lies at least about 3.5˚ A apart from
the wall of the hydrophobic pocket (the closest residue is
Ala250) and at more than 7˚ A from Ile255. Compared to
the BzlSA bound structure, the conformation of the latter
residue is substantially shiftedas measured by the Cα-Cβ-
Figure 4: Fit of a competitive inhibition model (continuous line-
squares, BzlSA; dashed line-triangles, citrate) to the experimental
Table 3: Kinetic parameters of BzlSA and citrate inhibition.
Cγ1-Cδ1 torsion angle: 160.3◦and −37.7◦for the BzlSA and
citrate CPA-bound forms, respectively. This rearrangement
to adapt the hydrophobic pocket to the features of the ligand
3.4. Kinetic Assays of CPA Ligands. Kinetic activity measure-
ments were carried out to determine the ability of citrate to
inhibit the CPA catalyzed reaction. The measurements were
made in parallel with BzlSA to provide a direct comparison
with this standard inhibitor. The nonlinear global fitting
of the experimental data to a competitive inhibition model
was excellent (Figure 4). Besides, the shape of the citrate
inhibition curve resembles that of BzlSA whose Ki is in
excellent agreement with values obtained in previous works
From Table 3, it is evident that citrate performs as
a very weak CPA inhibition compared to BzlSA, a likely
consequence of the presence of the aromatic moiety in the
latter. BzlSA was designed as a by-product analogue, that
is, it retains features of the cleaved products of the CPA
catalytic reaction  in such as way that the aromatic
moiety docks into the hydrophobic pocket of the enzyme,
displacing water molecules at the bottom of the active
site cleft. This displacement, together with the movement
of Tyr248, substantially facilitate the enzymatic activity by
creating an overall hydrophobic environment. It is well
documented that amino acid substrates and products are
very weak CPA inhibitors. For example, L-Phe is, like it has
been calculated here for citrate, a 5mM CPA inhibitor .
Although citrate would be easily displaced by the substrate
AAFP when competing for binding to CPA in the in vitro
experimental assays, a completely different scenario may be
possible in vivo such that citrate could be equally well suited
Citric acid has intestinal absorption enhancing effects and is
widely used in drug formulations. Recent evidence demon-
strated that the citrate salt of chitosan has a drug penetration
enhancement and that this effect is linked to the inhibition
of metal-dependent enzymes like leucine aminopeptidase
(a membrane protease) and the luminal protease CPA [44,
45]. In line with these observations, we report further
structural and kinetic evidence of the interaction between
CPA and citrate, in which the organic anion chelates the zinc
cation at the active site of the enzyme. The citrate mode
of binding resembles that of the peptidomimetic substrate
analogue benzylsuccinic acid, which also coincides with
citrate in being a competitive CPA inhibitor. The Ki value
for citrate is much weaker than that of benzylsuccinic acid
but comparatively similar to that of simple amino acids,
suggesting that citrate would compete for the in vivo binding
of zinc at the active site of intestinal enzymes.
There is a wide range of digestive protease inhibitors
with potential as peptide drug-protecting agents that, like
commonly used excipients, may be associated with some
drawbacks as elevated costs or risk of toxic side effects
[46, 47]. Efforts to combine a protease inhibitor and a drug
permeation enhancer in one single molecule to increase
the stability and absorption of peptide or protein drugs
are an increasingly investigated matter . As recently
reported , citrate, a commonly used excipient, may be
an example of such type of molecules. The analysis of the
newly determined carboxypeptidase-Zn2+-citrate complex
structure might help in the structure-guided design of newer
drug protecting compounds targeted to metal-dependent
Financial support from the Ministerio de Ciencia e Inno-
vaci´ on, Spain (Grant BIO2007-68046) and Generalitat de
Catalunya (Grant 2005SGR-1037) is gratefully acknowl-
staff at the EMBL (European Molecular Biology Laboratory),
Outstation at DESY (Deutsches Elektronen Synchrotron),
Hamburg, Germany, for their assistance in X-ray data
Bode, and M. Cygler, Eds., pp. 176–189, John Wiley & Sons,
Chichester, UK, 2004.
 R. H. Erickson and Y. S. Kim, “Digestion and absorption of
dietary protein,” Annual Review of Medicine, vol. 41, pp. 133–
 J. Berger and B. Olds Schneeman, “Stimulation of bile-
pancreatic zinc, protein and carboxypeptidase secretion in
response to various proteins in the rat,” Journal of Nutrition,
vol. 116, no. 2, pp. 265–272, 1986.
 J. Berger and B. O. Schneeman, “Intestinal zinc and car-
boxypeptidase A and B activity in response to consumption
of test meals containing various proteins by rats,” Journal of
Nutrition, vol. 118, no. 6, pp. 723–728, 1988.
 A. Ibarz, A. Garv´ ın, S. Garza, and J. Pag´ an, “Toxic effect
of melanoidins from glucose-asparagine on trypsin activity,”
Food and Chemical Toxicology, vol. 47, no. 8, pp. 2071–2075,
 C. J. Martin and W. J. Evans, “Phytic acid-enhanced metal ion
exchange reactions: the effect on carboxypeptidase A1,” Jour-
nal of Inorganic Biochemistry, vol. 35, no. 4, pp. 267–288, 1989.
 J. Parellada, G. Su´ arez, and M. Guinea, “Inhibition of
zinc metallopeptidases by flavonoids and related phenolic
compounds: structure-activity relationships,” Journal of
Enzyme Inhibition, vol. 13, no. 5, pp. 347–359, 1998.
 M. M. Mart´ ınez-Montemayor, G. M. Hill, N. E. Raney et al.,
“Gene expression profiling in hepatic tissue of newly weaned
pigs fed pharmacological zinc and phytase supplemented
diets,” BMC Genomics, vol. 9, article 421, 2008.
 M. A. Hilal, C. T. Ung, S. Westlake, and C. D. Johnson,
pancreatic acinar injury, but not L-selectin, a marker of
neutrophil activation, predicts severity of acute pancreatitis,”
Journal of Gastroenterology and Hepatology, vol. 22, no. 3, pp.
 S. Regn´ er, S. Appelros, C. Hjalmarsson, J. Manjer, J. Sadic, and
A. Borgstr¨ om, “Monocyte chemoattractant protein 1, active
carboxypeptidase B and CAPAP at hospital admission
are predictive markers for severe acute pancreatitis,”
Pancreatology, vol. 8, no. 1, pp. 42–49, 2008.
 J. S´ aez, J. Mart´ ınez, C. Trigo et al., “Clinical value of rapid
urine trypsinogen-2 test strip, urinary trypsinogen activation
peptide, and serum and urinary activation peptide of
carboxypeptidase B in acute pancreatitis,” World Journal of
Gastroenterology, vol. 11, no. 46, pp. 7261–7265, 2005.
 S. Matsugi, T. Hamada, N. Shioi, T. Tanaka, T. Kumada, and S.
Satomura, “Serum carboxypeptidase A activity as a biomarker
for early-stage pancreatic carcinoma,” Clinica Chimica Acta,
vol. 378, no. 1-2, pp. 147–153, 2007.
 F. Fialka, R. M. Gruber, R. Hitt et al., “CPA6, FMO2, LGI1,
SIAT1 and TNC are differentially expressed in early- and
late-stage oral squamous cell carcinoma—a pilot study,” Oral
Oncology, vol. 44, no. 10, pp. 941–948, 2008.
 P. L. Ross, I. Cheng, X. Liu et al., “Carboxypeptidase 4 gene
variants and early-onset intermediate-to-high risk prostate
cancer,” BMC Cancer, vol. 9, article 69, 2009.
tosis,” Proceedings of the National Academy of Sciences of the
 J. D. Johnson, “Proteomic identification of carboxypeptidase
E connects lipid-induced β-cell apoptosis and dysfunction in
type 2 diabetes,” Cell Cycle, vol. 8, no. 1, pp. 38–42, 2009.
 D. A. Tregouet, R. Schnabel, M. C. Alessi et al., “Activated
with the risk of cardiovascular death in patients with coronary
artery disease: the Athero Gene study,” Journal of Thrombosis
and Haemostasis, vol. 7, no. 1, pp. 49–57, 2009.
 L. L. Leung, T. Myles, T. Nishimura, J. J. Song, and W. H.
Robinson, “Regulation of tissue inflammation by thrombin-
Immunology, vol. 45, no. 16, pp. 4080–4083, 2008.
 C. Schmuck, “How to improve guanidinium cations for oxo-
anion binding in aqueous solution?. The design of artificial
peptide receptors,” Coordination Chemistry Reviews, vol. 250,
no. 23-24, pp. 3053–3067, 2006.
 D. M. Hayes and P. A. Kollman, “Electrostatic potentials of
proteins. 1. Carboxypeptidase A,” Journal of the American
Chemical Society, vol. 98, no. 11, pp. 3335–3345, 1976.
 L. Banci, I. Bertini, and G. La Penna, “The enzymatic mech-
anism of carboxypeptidase: a molecular dynamics study,”
Proteins: Structure, Function and Genetics, vol. 18, no. 2, pp.
 K. Zhang, J. Dong, and D. S. Auld, “A time-resolved X-Ray
absorption fine structure study of substrate hydrolysis by
carboxypeptidase A,” Physica B (Amsterdam), vol. 208 & 209,
no. 1–4, pp. 719–721, 1995.
 S.´Alvarez-Santos,`A. Gonz´ alez-Lafont, J. M. Lluch, B. Oliva,
and F. X. Avil´ es, “Theoretical study of the role of arginine
127 in the water-promoted mechanism of peptide cleavage by
carboxypeptidase A,” New Journal of Chemistry, vol. 22, no. 4,
pp. 319–325, 1998.
role of Tyr248 probed by mutant bovine carboxypeptidase A:
insight into the catalytic mechanism of carboxypeptidase A,”
Biochemistry, vol. 40, no. 34, pp. 10197–10203, 2001.
 D. Xu and H. Guo, “Quantum mechanical/molecular
mechanical and density functional theory studies of a
prototypical zinc peptidase (carboxypeptidase A) suggest a
Chemical Society, vol. 131, no. 28, pp. 9780–9788, 2009.
 D. H. Kim, “Chemistry-based design of inhibitors for car-
boxypeptidase A,” Current Topics in Medicinal Chemistry, vol.
4, no. 12, pp. 1217–1226, 2004.
 M. Adler, B. Buckman, J. Bryant et al., “Structures of potent
selective peptide mimetics bound to carboxypeptidase B,”
Acta Crystallographica. Section D: Biological Crystallography,
vol. 64, no. 2, pp. 149–157, 2008.
 M. E. Bunnage and D. R. Owen, “TAFIa inhibitors in the
treatment of thrombosis,” Current Opinion in Drug Discovery
and Development, vol. 11, no. 4, pp. 480–486, 2008.
 I. Bravo-Osuna, C. Vauthier, A. Farabollini, G. Millotti, and
G. Ponchel, “Effect of chitosan and thiolated chitosan coating
on the inhibition behaviour of PIBCA nanoparticles against
vol. 10, no. 8, pp. 1293–1301, 2008.
 R. Rink, A. Arkema-Meter, I. Baudoin et al., “To protect
peptide pharmaceuticals against peptidases,” Journal of
Pharmacological and Toxicological Methods, vol. 61, no. 2, pp.
 D. Fern´ andez, S. Testero, J. Vendrell, F. X. Avil´ es, and S.
Mobashery, “The X-ray structure of carboxypeptidase a
inhibited by a thiirane mechanism-based ihibitor,” Chemical
Biology and Drug Design, vol. 75, no. 1, pp. 29–34, 2010.
 G. V. Odell, P. C. Ferry, L. M. Vick et al., “Citrate inhibition
of snake venom proteases,” Toxicon, vol. 36, no. 12, pp.
 M. C. Bonferoni, G. Sandri, S. Rossi, F. Ferrari, S. Gibin, and
C. Caramella, “Chitosan citrate as multifunctional polymer
for vaginal delivery. Evaluation of penetration enhancement
and peptidase inhibition properties,” European Journal of
Pharmaceutical Sciences, vol. 33, no. 2, pp. 166–176, 2008.
 Collaborative Computational Project number 4, “The
CCP4 suite: programs for protein crystallography,” Acta
Crystallographica. Section D: Biological Crystallography, vol.
50, pp. 760–776, 1994.
 R. J. Read, “Pushing the boundaries of molecular replacement
with maximum likelihood,” Acta Crystallographica. Section
D Biological Crystallography, vol. 57, no. 10, pp. 1373–1382,
 G. N. Murshudov, A. A. Vagin, and E. J. Dodson, “Refinement
of macromolecular structures by the maximum-likelihood
Crystallography, vol. 53, no. 3, pp. 240–255, 1997.
 I. W. Davis, L. W. Murray, J. S. Richardson, and D. C.
Richardson, “MolProbity: structure validation and all-atom
contact analysis for nucleic acids and their complexes,” Nucleic
Acids Research, vol. 32, pp. W615–W619, 2004.
 R. A. Laskowski, M. W. MacArthur, E. Hutchinson, and
J. M. Thornton, “PROCHECK: a program to check the
stereochemical quality of protein structures,” Journal of
Applied Crystallography, vol. 26, pp. 283–291, 1993.
 P. Emsley and K. Cowtan, “Coot: model-building tools for
molecular graphics,” Acta Crystallographica. Section D: Biolog-
ical Crystallography, vol. 60, no. 12, pp. 2126–2132, 2004.
 Y. Cheng and W. H. Prusoff, “Relationship between the
inhibition constant (K1) and the concentration of inhibitor
which causes 50 per cent inhibition (I50) of an enzymatic
reaction,” Biochemical Pharmacology, vol. 22, no. 23, pp.
 D. Fern´ andez, E. Boix, I. Pallar` es, F. X. Avil´ es, and J. Vendrell,
“Analysis of a new crystal form of procarboxypeptidase B:
further insights into the catalytic mechanism,” Biopolymers,
vol. 93, no. 2, pp. 178–185, 2010.
 S. Firth-Clark, S. B. Kirton, H. M. G. Willems, and A.
Williams, “De novo ligand design to partially flexible active
sites: application of the ReFlex algorithm to carboxypeptidase
A, acetylcholinesterase, and the estrogen receptor,” Journal
of Chemical Information and Modeling, vol. 48, no. 2, pp.
 L. D. Byers and R. Wolfenden, “A potent reversible inhibitor
of carboxypeptidase A,” The Journal of Biological Chemistry,
vol. 247, no. 2, pp. 606–608, 1972.
 R. E. Galardy and Z. P. Kortylewicz, “Inhibition of
carboxypeptidase A by aldehyde and ketone substrate
analogues,” Biochemistry, vol. 23, no. 9, pp. 2083–2087, 1984.
 M. C. Bonferoni, G. Sandri, S. Rossi, F. Ferrari, and C. Cara-
mella, “Chitosan and its salts for mucosal and transmucosal
delivery,” Expert Opinion on Drug Delivery, vol. 6, no. 9, pp.
 T. Kean, S. Roth, and M. Thanou, “Trimethylated chitosans as
non-viral gene delivery vectors: cytotoxicity and transfection
efficiency,” Journal of Controlled Release, vol. 103, no. 3, pp.
 M. G. Ursino, E. Poluzzi, C. Caramella, and F. De Ponti,
“Excipients in medicinal products used in gastroenterology
as a possible cause of side effects,” Regulatory Toxicology and
Pharmacology, vol. 60, no. 1, pp. 93–105, 2011.
Section D: Biological
 M. D. Del Curto, A. Maroni, L. Palugan, L. Zema, A. Gaz-
zaniga, and M. E. Sangalli, “Oral delivery system for two-pulse
colonic release of protein drugs and protease inhibitor/ ab-
sorption enhancer compounds,” Journal of Pharmaceutical
Sciences, vol. 100, no. 8, pp. 3251–3259, 2011.
 M. P. Wentland, S. Raza, and Y. Gao, “96-Well plate colori-
metric assay for Ki determination of (±)-2-Benzylsuccinic
acid, an inhibitor of carboxypeptidase A. A laboratory experi-
ment in drug discovery,” Journal of Chemical Education, vol.
81, no. 3, pp. 398–400, 2004.