Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr607 residue

Article abstract of DOI:10.1016/j.abb.2010.05.025

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz=ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X=different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.

Full text of DOI:10.1016/j.abb.2010.05.025

Archives of Biochemistry and Biophysics 500 (2010) 131–136
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA)
and the role of the Tyr607 residue
Ricardo Z. Lorenzon a,1, Carlos E.L. Cunha a,1, Marcelo F. Marcondes a, Maurício F.M. Machado a,
a
Maria A. Juliano a, Vitor Oliveira a, Luiz R. Travassos b, Thaysa Paschoalin a, , Adriana K. Carmona
*
a Departamento de Biofísica, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP 04044-020, Brazil
b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, São Paulo, SP 04023-062, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2010
and in revised form 19 May 2010
Available online 27 May 2010
Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and struc-
tural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three
enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In
OpdA, the flexible loop is formed by residues 600–614 (600SHIFAGGYAAGYYSY614). Modeling studies indi-
cated that in OpdA the Tyr607 residue might be involved in the recognition of the substrate with a key role
in catalysis. Two mutants were constructed replacing Tyr607 by Phe (Y607F) or Ala (Y607A) and the influ-
ence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz–
GXSPFRQ–EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenedi-
amine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT)
and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only
on the X–S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X–S and the P–F
bonds. The kinetic parameters showed the importance of Tyr607 in OpdA catalytic activity as its substitu-
tion promoted a decrease in the kcat/Km value of about 100-fold with Y607F mutant and 1000-fold with
Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluores-
cence analysis. Our results indicate that the OpdA Tyr607 residue plays an important role in the enzyme-
substrate interaction and in the hydrolytic activity.
Keywords:
M3A subfamily
E. coli oligopeptididase A
Site-directed mutagenesis
FRET substrates
Ó 2010 Elsevier Inc. All rights reserved.
Introduction
boxypeptidase (Dcp),2 anenzymethatcloselyresemblesthemamma-
lian angiotensin I-converting enzyme [9–11].
Proteolysis is an essential process that controls the protein bal-
ance regulating the life cycles in all cells, including bacteria [1–3].
Several bacterial peptidases are described to play an important role
in proteindegradationgeneratingpeptide fragmentsthatare further
hydrolyzed by oligopeptidases to smaller peptides and amino acids.
Among them is the oligopeptidase A (OpdA), a zinc-dependent en-
zyme of the M3A subfamily described in Salmonella typhimurium
and Escherichia coli [4–6]. The M3A subfamily is characterized by a
common active site sequence motif (HEXXH) which is highly con-
served in several metallopeptidases [7,8]. A search in E. coli trans-
lated genome for this motif indicated the presence of two
peptidases belonging to the M3 family, OpdA and dipeptidyl car-
Thimet oligopeptidase (TOP) and neurolysin (NEL) are examples
of mammalian metallopeptidases from M3A subfamily. These two
enzymes share about 60% sequence identity, have similar tissue
distribution [12,13] and very similar substrate specificity [12,14–
16]. OpdA is the bacterial member of the M3A subfamily with
680 amino acid residues and 77.1 kDa molecular mass [5]. It has
been suggested that OpdA could be a general protease that partic-
ipates in multiple catabolic pathways in E. coli due to its ability to
hydrolyze small peptides of a broad size range [17]. Recently, it
was shown that the recombinant enzyme has the ability to hydro-
lyze the same synthetic substrates cleaved by TOP and NEL, besides
bioactive peptides like bradykinin and neurotensin [8]. Further-
more, the complete inhibition of OpdA by the TOP inhibitor JA-2
(N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate)
and partially by the NEL inhibitor Pro-Ile [18], give support to the
2
Abbreviations used: Abz, ortho-aminobenzoic acid; Dcp, dipeptidyl carboxypepti-
dase; EDDnp, N-[2,4-dinitrophenyl]ethylenediamine; FRET, fluorescence resonance
energy transfer; MALDI-TOF, matrix assisted laser desorption ionization–time of
flight; MS, mass spectroscopy; NEL, neurolisin; TOP, thimet-oligopeptidase; OpdA
WT, wild type OpdA.
* Corresponding author. Fax: +55 11 55755877.
E-mail address: tpaschoalin@unifesp.br (T. Paschoalin).
1
These authors contributed equally to this study.
0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2010.05.025

132
R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136
similar structural/functional activity between the mammalian and
the bacterial oligopeptidase [8].
pET vector, containing cDNA encoding the desired protein (GE
Healthcare, Chalfont St. Giles, UK), as previously described [27]. Re-
combinant proteins were purified to homogeneity by affinity chro-
An important characteristic of M3A family members is that the
catalytic site is located in a deep channel that provides access only
to short peptides [19,20]. The broad substrate specificity displayed
by TOP and NEL are explained in part by the presence of a flexible
loop lined with the enzyme binding site of these peptidases
[15,16,20–23]. Site-directed mutagenesis in specific amino acid
residues in the conserved flexible loop have demonstrated the
importance of this region for the enzyme-substrate interaction
and showed that the loop flexibility is essential for catalysis [22–
25]. Such flexible loop has 13 amino acid residues in which TOP
and NEL share 12 of them, with only one difference at the ninth po-
sition where NEL has a Gly residue instead of an Ala present in TOP.
Interestingly, OpdA flexible loop differs from TOP and NEL, but the
three enzymes have four conserved Tyr residues. Comparison of
the crystallographic structures of TOP [20] and NEL [19] with that
of E. coli dipeptidyl carboxypeptidase (Dcp) [26] suggests that the
bacterial members of M3 family may also undergo a large hinge
movement upon substrate or inhibitor binding closing their deep
channels around the substrate or inhibitor.
In the present work we carried out OpdA modeling studies
based on crystal structures of homologous enzymes [20,26]. The
result indicated that the OpdA Tyr607, present in the flexible loop
formed by the residues 600–614 (600SHIFAGGYAAGYYSY614), might
be involved in the recognition of the substrate having a key role in
the enzyme selectivity. To address this possibility, we constructed
two mutants where the Tyr607 residue was replaced by Phe (Y607F)
or Ala (Y607A) and studied the influence of this site-directed muta-
genesis in the enzyme catalytic activity and protein folding in or-
der to reveal the importance of the aromatic ring and/or of the
hydroxyl group on the enzyme activity. Kinetic studies with OpdA
WT, and both mutants were carried out using fluorescence reso-
nance energy transfer (FRET) bradykinin-related peptides as sub-
strates. In addition, CD and intrinsic fluorescence analysis were
performed with OpdA WT enzyme and both mutant enzymes.
Our results suggest that both aromatic ring and hydroxyl group
are crucial for substrate catalysis.
matography on
a nickel-Sepharose column (GE Healthcare,
Chalfont St. Giles, UK). After purification, recombinant proteins
were analyzed by SDS–PAGE followed by staining with Coomassie
Blue. Protein batches with a homogeneity >95% were stored at 4 °C
and used in all subsequent analyses.
Circular dichroism (CD) and thermal stability
Far UV-CD spectra were recorded on a Jasco J-810 spectropolar-
imeter with a Peltier system for controlling cell temperature. The
system was routinely calibrated with an aqueous solution of twice
crystallized d-10 camphorsulfonic acid. Ellipticity was recorded as
the mean residue molar ellipticity [h] (deg cm2 dmolꢀ1). The spec-
trometer conditions typically included a sensitivity of 100 mdeg,
resolution of 0.5 nm, response time of 4 s, scan rate of 20 nm/
min, and four accumulations at 37 °C. The kinetics of denaturation
of the OpdA WT and mutant peptidases (Y607F and Y607A) were
monitored by following the intrinsic fluorescence during incuba-
tion at 50 °C. The buffer used in the assays was 50 mM Tris–HCl,
pH 7.4. The intrinsic fluorescence was monitored continuously
(see kinetic assays) with excitation at 280 nm and emission at
330 nm (2.5 nm slit width in both cases).
Peptide synthesis
TheFRET peptidescontainingthe fluorescentgroup ortho-amino-
benzoic acid (Abz) and the acceptor N-[2,4-dinitrophenyl]ethylene-
diamine (EDDnp) were synthesized at the Biophysics Department of
Federal University of São Paulo (São Paulo–Brazil). The synthesis
used the Fmoc procedure in an automated bench-top simultaneous
multiple solid-phase peptide synthesizer (PSSM 8 system from Shi-
madzu, Tokyo, Japan) as described elsewhere [28]. The final depro-
tected peptides were purified by semi-preparative HPLC using an
Econosil C18 column (10
lm, 22.5 ꢁ 250 mm) and a two-solvents
system: (A) trifluoroacetic acid (TFA)/H2O (1:1000, v/v) and (B)
TFA/acetonitrile (ACN)/H2O (1:90:10, v/v). The column was eluted
at a flow rate of 5 mL/min with a 10 (or 30)% to 50 (or 60)% gradient
of solvent B over 30 or 45 min. Analytical HPLC was run using a bin-
ary HPLC system from Shimadzu fitted with SPD-10AV Shimadzu
UV–vis detector and Shimadzu RF-535 fluorescence detector. The
Materials and methods
Protein model generation
system was coupled to an Ultrasphere C18 column (5 lm,
The 3D model of OpdA was created using the FASTA sequence of
OpdA itself (GI 16131370), the crystal structures of TOP (1S4B,
2O36), Dcp (1Y79), NEL (2O3E, 1I1I) and ACE2 (1R42, 1R4L)
through the Swiss Model Algorithm (http://swissmodel.exp-
asy.org/). These structures were checked for aberrations using
two online validation servers: WHATIF (http://swift.cmbi.kun.nl/
whatif/) and PRO_CHECK (http://www.ebi.ac.uk/thornton-srv/soft-
ware/PROCHECK/). Finally those structures were refined using
WHAT_CHECK (http://swift.cmbi.ru.nl/gv/whatcheck/), and then
they were validated all over again. After this repetitive procedure
two models were obtained: one of OpdA in its ‘‘open” state, and an-
other of OpdA in its ‘‘closed” state.
4.6 ꢁ 150 mm) that was eluted with solvent systems A and B at a
flow rate of 1 mL/min and 10–80% gradient of B over 20 min. The elu-
tion profile of the peptides was monitored by the absorbance at
220 nm and by the fluorescence emission at 420 nm following exci-
tation at 320 nm. The molecular mass and purity of the synthesized
peptides (94% or higher) were checked by matrix assisted laser
desorption ionization/time of flight (MALDI-TOF) mass spectrome-
try (TofSpec-E, Micromass, Manchester, UK) and peptide sequencing
with a PPSQ-23 protein sequencer (Shimadzu, Tokyo, Japan). Stock
solutionsof thepeptideswere prepared inDMSO, and theconcentra-
tions were measured spectrophotometrically using the molar
extinction coefficient of 17,300 Mꢀ1cmꢀ1 at 365 nm.
Site-directed mutagenesis and protein expression and purification
Kinetic assays
The QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA, USA) was used to introduce punctual mutations in
E. coli OpdA WT cDNAs, as previously described [27]. The specific
mutations were all confirmed by DNA sequencing [27] and identi-
fied as OpdA Y607F and OpdA Y607A, here named Y607F and
Y607A, respectively. The enzymes OpdA WT, Y607F and Y607A
The hydrolysis of fluorescence-quenched bradykinin derivatives
by OpdA WT and by Y607F and Y607A mutants was continuously
monitored in a Hitachi F-2000 fluorimeter(Tokyo, Japan), measuring
the fluorescence at kem = 420 nm and kex = 320 nm. Peptides with
general sequence Abz–GXSPFRQ–EDDnp (Abz = ortho-aminoben-
zoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine), where
X represents Ala, Gly, Leu, Phe, Tyr, Ser, Gln, Asn, Glu, Asp, His, Arg,
were expressed in E. coli DH5a using the pHis3 plasmid, a modified

R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136
133
Lys and Pro were tested. The kinetic parameters of peptide hydroly-
sis were determined at 37 °C in 50 mM Tris–HCl buffer, pH 7.4. The
reaction was monitored continuously based on the fluorescence of
the released product. The rate of fluorescence increase was con-
verted in lmoles of substrate hydrolyzed per minute based on the
fluorescence curves of standard peptide solutions before and after
total enzymatic hydrolysis. The enzyme concentration for initial rate
determinations was chosen so that <5% of the substrate was hydro-
lyzed. The inner-filter effect was corrected using an empirical equa-
tion [29]. The data were analyzed and equations were fitted using
Grafit program [30].
Determination of peptide cleavage sites
The hydrolysis of the FRET peptides was analyzed by LC/ESI-MS
using a Shimadzu apparatus (Shimadzu Corporation, Tokyo, Japan),
model 2010 with a SPD-20A Shimadzu UV/vis detector and RF-
10AXL fluorescence detector coupled with an Ultrasphere C-18 col-
umn (5
l
m, 4.6 ꢁ 250 mm). A linear gradient of 10–80% of solvent
B was run for 20 min after 8 min of isocratic flow. Solvent A: 0.1%
TFA in H2O; solvent B: 0.1% TFA in CH3CN/H2O (75:25).
The effect of ionic strength and pH on catalytic activity
The pH dependence on Abz–GFSPFRQ–EDDnp hydrolysis was
determined under pseudo-first-order conditions, over a pH range
of 5.5–10.0. Determinations were carried out at 37 °C using a
four-component buffer comprised of 25 mM glycine, 25 mM acetic
acid, 25 mM Mes and 75 mM Tris [31]. Eq. (1), was used in the non-
linear regression analysis using Grafit program [30].
1Þ
2Þ
Limit1 þ Limit2 ꢂ 10ðpHꢀpK Limit2 þ Limit3 ꢂ 10ðpHꢀpK
K ¼
ð1Þ
10ðpHꢀpK Þ þ 1
10ðpHꢀpK Þ þ 1
1
2
Eq. (1) fits data when the pH–activity profile depends upon two
ionizing groups (double pKa) and does not assume that the activity
is zero at high pH values. Limit1 represents the limit of the acid
limb (low pH), Limit2 is the pH-independent maximum rate con-
stant, K1 and K2 are the dissociation constants of a catalytically
competent base and acid, respectively, and Limit3 is the limit of
the alkaline limb (high pH) k = kcat/Km.
The influence of NaCl on the catalytic activity of OpdA WT,
Y607F and Y607A was investigated using Abz–GFSPFRQ–EDDnp
as substrate, at 37 °C, in 50 mM Tris–HCl, pH 7.4, over a NaCl range
of 0–2 M. The enzymatic activity was followed as described above.
Fig. 1. Crystal structure of Dcp (yellow), the generated model of OpdA (red) in its
open (A) and closed (B) conformation, with the zinc ion (gray) coordinated by two
histidines and one glutamic acid residue. In black, modeled peptides sitting in the
active site, with a larger emphasis on the P1 residue. (A) the Tyr607 in the flexible
loop (green) is shown to be far apart the active site (10 Å) and the residue sitting on
the P1 position (6 Å); (B) OpdA in closed conformation, Tyr607 is much closer to the
active site (5 Å) and even closer to the residue sitting on P1 (4 Å) as compared to the
open conformation. (For interpretation of the references to color in this figure, the
reader is referred to the web version of the article.)
ever, when OpdA assumes the closed conformation, Tyr607 moves
to a position closer to the active site and even closer to the residue
on P1 (4 Å). These modeling data suggested that the residue Tyr607
is important for the OpdA catalytic activity as demonstrated for the
corresponding Tyr residues in other members of M3A family [23].
In order to confirm our modeling finding two mutants were
constructed substituting Tyr607 by Phe (Y607F) and Ala (Y607A).
Results and discussion
Modeling studies
The 3D structural model of TOP [20], NEL [19] and Dcp [26] re-
vealed that some residues undergo a major conformation change,
mainly those directly involved the catalytic reaction itself. However,
other residues placed far from the catalytic site of these enzymes
alsounderwentsignificantdistortion, mainly those present in a loop,
which is thought to have a role on substrate selectivity. This is the
case of residue Tyr605 in TOP and Tyr606 in NEL [23]. This fact led us
to investigate if the corresponding Tyr607 in OpdA is involved in sub-
strate recognition, which is coherent with the role of this conserved
flexible loop present in all members of M3A family.
A comparative modeling study of OpdA in open conformation
(Fig. 1A) and in closed conformation (Fig. 1B) indicated that there
is a great displacement of the loop, moving the Tyr607 towards
the active site when OpdA assumes its closed conformation. In
Fig. 1A, Tyr607 is shown to be far apart the active site (10 Å) and
also far from the residue sitting in P1 position (6 Å). In Fig. 1B, how-
Enzyme expression, conformational and thermal stability studies
OpdA WT and the mutants Y607F and Y607A were expressed in
E. coli and purified in a nickel-Sepharose column. The homogeneity
of the purified proteins (95% purity) was assessed by polyacryl-
amide gel electrophoresis after staining with Coomassie Blue (data
not shown).
To further characterize the expressed enzymes, Far-UV-CD
spectra was performed showing that OpdA and its mutants showed
predominant
a-helical structures (Fig. 2A). The spectra obtained
for both Y607F and Y607A mutants were similar to the spectrum
for the wild-type enzyme. In the same way, no significant

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R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136
NEL [23]. The peptide Abz–GKSPFRQ–EDDnp was hydrolyzed by
Y607F on both peptide bonds X–Ser (42.3%) and Pro–Phe (57.7%).
OpdA Y607A mutant cleaved peptides Abz–GYSPFRQ–EDDnp,
Abz–GRSPFRQ–EDDnp, Abz–GHSPFRQ–EDDnp and Abz–GFSPFRQ–
EDDnp on the X–Ser bond only. Peptides containing the residues
Gly, Leu and Ser on the X position were hydrolyzed on the Pro–Phe
bond. The substrates Abz–GASPFRQ–EDDnp (Ala–Ser = 70% and
Pro–Phe = 30%); Abz–GLSPFRQ–EDDnp (Leu–Ser = 50% and Pro–
Phe = 50%); Abz–GQSPFRQ–EDDnp (Gln–Ser = 50% and Pro–Phe =
50%); Abz–GNSPFRQ–EDDnp (Asn–Ser = 58% and Pro–Phe = 42%);
and Abz–GKSPFRQ–EDDnp (Lys–Ser = 40% and Pro–Phe = 60%) were
cleaved on both X–Ser and Pro–Phe bonds. Moreover, the peptides
containing the negatively charged amino acids Asp and Glu, as well
as Pro in the X position were resistant to hydrolysis even at high con-
centration of enzyme. Such different behavior according with the
substrate including changes of the cleavage site where also observed
with TOP Y605F, TOP Y605A [23,24], NEL Y606F and NEL 606A [23].
This observation suggests a similar role for OpdA Y607 residue in
substrate hydrolysis to that of Y605/Y606 of TOP/NEL [23,24].
The kinetic parameters obtained for the hydrolysis of Abz–
GXSPFRQ–EDDnp derivatives by OpdA and Y607F and Y607A mu-
tants indicated that the OpdA WT has high affinity for the basic
residue Arg at P1 position. Substrate Abz–GRSPFRQ–EDDnp was
hydrolyzed by OpdA at the highest catalytic efficiency in all
peptide series (kcat/Km = 10,358 mMꢀ1 sꢀ1). Surprisingly, the
peptides Abz–GHSPFRQ–EDDnp (kcat/Km = 1910 mMꢀ1 sꢀ1
) and
Abz–GKSPFRQ–EDDnp (kcat/Km = 1095 mMꢀ1 sꢀ1) that contain in
P1 the positively charged residues His and Lys, respectively, were
not as suitable for hydrolysis as Abz–GRSPFRQ–EDDnp. The hydro-
phobic aromatic residues Phe (kcat/Km = 1584 mMꢀ1 sꢀ1) and Tyr
(kcat/Km = 2191 mMꢀ1 sꢀ1) were also well accepted by the S1 sub-
site. In contrast, peptides containing Asp and Glu in the P1 position
were poorly hydrolyzed by OpdA, while neutral polar residues,
Asn, Ser and Gln, and polar residues, Pro, Gly and Leu, did not show
any important interaction with the active site.
The kinetic parameters determined for Y607F and Y607A mu-
tants demonstrated that the presence the Tyr607 residue is essen-
tial for the OpdA catalytic activity. Table 1 and Fig. 3 show a
decrease in catalytic activity of about 100-fold for Y607F mutant
and 1000-fold for Y607A mutant. These lower kcat/Km values are
in response mainly to the drastic kcat decrease. Peptides containing
Pro, Asp and Glu residues on the X position were resistant to
Fig. 2. (A) Far-UV-CD spectra of OpdA WT and Y607F and Y607A mutants. (B)
Intrinsic fluorescence (kex = 280 nm and kem = 330 nm) of OpdA WT (s), Y607F (d)
and Y607A (h) during incubation at 50 °C.
difference was observed in the intrinsic fluorescence (Fig. 2B) of
the mutant peptidases when compared with the wild-type en-
zyme. Therefore, the site-directed mutagenesis of the Tyr607 resi-
due did not promote significant changes in the secondary
structure of the proteins.
hydrolysis up to 2.1 lM enzyme concentration. This confirms the
importance, not only of the hydroxyl group, but mainly of the aro-
matic ring on the 607th position for OpdA activity. Both mutant en-
zymes showed their highest kcat/Km values with peptides bearing
aromatic amino acids on the P1 position, especially the Tyr residue.
Hydrolysis of synthetic fluorogenic substrates
To compare the catalytic activity of OpdA WT and that of Y607F
and Y607A mutants, FRET peptides based on the general structure
Abz–GXSPFRQ–EDDnp (X = Ala, Pro, Leu, Gly, Phe, Tyr, Ser, Asn,
Gln, Lys, Arg, His, Glu or Asp) were used. The leader peptide of
the series, Abz–GFSPFRQ–EDDnp, contains six C-terminal amino
acids of bradykinin (RPPGFSPFR) which is as an efficient substrate
for OpdA [8]. The kinetic parameters determined and the cleavage
sites are shown in Table 1. The substitution of OpdA Tyr607 residue
by Phe or Ala significantly altered the kinetic parameters for the
hydrolysis of Abz–GXSPFRQ–EDDnp derivatives in comparison
with the OpdA WT. In addition, the OpdA mutants showed differ-
ent cleavage sites indicating that the Tyr607 residue is important
to define the P1 residue in the substrate hydrolysis.
OpdA WT hydrolyzed all the substrates on the X–Ser bond only.
However, a different pattern of cleavage was observed for the mu-
tants. Although OpdA mutant Y607F cleaved most of the peptides
on the X–Ser bond, the peptides Abz–GPSPFRQ–EDDnp, Abz–
GGSPFRQ–EDDnp, Abz–GESPFRQ–EDDnp and Abz–GDSPFRQ–
EDDnp were hydrolyzed on the Pro–Phe bond, just like TOP and
Influence of NaCl and pH on OpdA WT, and Y607F and Y607A mutant
activities
The influence of NaCl concentration on Abz–GFSPFRQ–EDDnp
hydrolysis by OpdA WT, Y607F and Y607A mutants was studied
over a range of 0–2.0 M NaCl. For each salt concentration, the
kcat/Km value was determined under pseudo-first-order conditions
using the fluorimetric assay described above (Fig. 4). Increase of
salt concentration rendered a significant rise of kcat/Km values for
both mutants mainly after the addition of 1 M NaCl. For Y607F
and Y607A, the kcat/Km values at high molar concentrations were
nearly 10 times higher than that with the OpdA WT enzyme. The
alterations seen in the salt curve are coherent with the idea that
this specific Tyr residue in the OpdA’s flexible loop is strongly re-
lated to the enzyme catalytic activity. For native OpdA however,
the increase in salt concentration and therefore the change in the
solvent did not significantly modify the enzyme activity.

R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136
135
Table 1
Kinetic parameters for the hydrolysis of FRET peptides derived from Abz–GXSPFRQ–EDDnp sequence by OpdA WT and Y607F and Y607A mutants.
X
OpdA WT
Y607F
Y607A
Km
(
lM)
kcat (sꢀ1
)
kcat/Km (mM s)ꢀ1
Km
(
lM)
kcat (sꢀ1
)
kcat/Km (mM s)ꢀ1
Km
(l
M)
kcat (sꢀ1
)
kcat/Km (mM s)ꢀ1
A
G
L
F
Y
S
Q
N
E
D
H
R
K
P
0.8
1.1
1.8
0.7
0.7
0.5
2.2
1.8
1.8
4.2
0.4
0.2
0.4
0.7
0.5
0.2
1.5
1.1
1.5
0.3
0.6
0.5
0.08
0.2
0.74
2.4
0.4
0.1
595
194
844
1584
2191
590
258
273
42
49
2.2
1.3
8.6
2.7
1.2
0.5
5
0.014
0.008
0.2
18
kcat/Km = 0.6 (X–S; P–F)*
6 (P–F)
2.1
36
55
7
9.4
8.9
6.1
6.5
10
0.02
0.3 (P–F)
0.032
0.025
0.064
0.003
4 (P–F)
4
10
0.1
0.065
0.004
0.05
0.02
kcat/Km = 0.25 (P–F)*
0.002
0.08
0.3 (P–F)
10
7
kcat/Km = 0.6 (X–S; P–F)*
kcat/Km = 0.2 (X–S; P–F)*
Resistant
2.6
4.7
4
0.7
0.5 (P–F)
20
49
Resistant
kcat/Km = 0.6*
1910
10,358
1095
159
0.034
1.7
0.005
3
kcat/Km = 7 (X–S; P–F)*
kcat/Km = 0.8 (P–F)*
kcat/Km = 0.5 (X–S; P–F)*
Resistant
a If unspecified, the peptides were hydrolyzed on the X–S bond; * indicates that kcat/Km was determined under pseudo first-order conditions. The errors were less than 10% for
any value.
Resistant = No hydrolysis occurred up to 2.1 lM enzyme concentration.
1
0.1
0.01
0.001
0.0001
A
G
L
F
Y
S
Q
N
E
D
H
R
K
P
Amino acid (X)
Fig. 3. Relative kcat/Km ratios for the hydrolysis of peptides derived from Abz–GXSPFRQ–EDDnp sequence by OpdA WT and Y607F and Y607A mutants. Black bars – Y607F/
OpdA WT, white bars – Y607A/OpdA WT.
Fig. 5. pH dependence for hydrolysis of Abz–GFSPFRQ–EDDnp by OpdA WT (d),
Fig. 4. NaCl influence in the hydrolysis of Abz–GFSPFRQ–EDDnp by OpdA WT
Y607F (s) and Y607A (j). The assays were performed as described in Material and
(s),Y607F (d) and Y607A (h). In the ratio (k/k0), k0 is the kcat/Km value obtained in
methods section. Values are presented as kcat/Km
.
50 mM Tris–HCl, pH 7.4, in the absence of salt and k is the kcat/Km value obtained in
a defined salt concentration.
The pH dependence on the hydrolysis of Abz–GFSPFRQ–EDDnp
by OpdA WT and by both mutants was determined over a pH range
of 5.5–10.0. Fig. 5 shows the pH-curve for the three enzymes, with
a pH optimum around 7.0 for the OpdA WT (pKe1 = 6.5 0.1;
pKe2 = 6.9 0.1) and Y607F (pKe1 = 6.0 0.1; pKe2 = 7.5 0.1), and
of 6.0 for the Y607A (pKe1 = 6.0 0.1; pKe2 = 6.3 0.1) mutant. Such
difference in the optimum pH parallels the different affinity with
the assayed substrates shown for the Y607A mutant enzyme. Thus,

136
R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136
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of Tyr607 and the importance of the contacts involving the aromatic
and hydroxyl groups of the tyrosine residue.
This difference on the catalytic properties and also the recogni-
tion of the correct cleavage site shows that important chemical
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tants Y607F and Y607A which cannot perform their hydrolytic
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Acknowledgments
This work was supported by Conselho Nacional de Desenvolvi-
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