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

Archives of Biochemistry and Biophysics 500 (2010) 131–136

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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, Maria A. Juliano a, Vitor Oliveira a, Luiz R. Travassos b, Thaysa Paschoalin a,*, Adriana K. Carmona a a b

Departamento de Biofísica, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP 04044-020, Brazil 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

Article history: Received 7 April 2010 and in revised form 19 May 2010 Available online 27 May 2010 Keywords: M3A subfamily E. coli oligopeptididase A Site-directed mutagenesis FRET substrates

a b s t r a c t 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 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 fluorescence analysis. Our results indicate that the OpdA Tyr607 residue plays an important role in the enzymesubstrate interaction and in the hydrolytic activity. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Proteolysis is an essential process that controls the protein balance regulating the life cycles in all cells, including bacteria [1–3]. Several bacterial peptidases are described to play an important role in protein degradation generating peptide fragments that are further hydrolyzed by oligopeptidases to smaller peptides and amino acids. Among them is the oligopeptidase A (OpdA), a zinc-dependent enzyme 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 conserved in several metallopeptidases [7,8]. A search in E. coli translated genome for this motif indicated the presence of two peptidases belonging to the M3 family, OpdA and dipeptidyl car-

* Corresponding author. Fax: +55 11 55755877. E-mail address: [email protected] (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

boxypeptidase (Dcp),2 an enzyme that closely resembles the mammalian angiotensin I-converting enzyme [9–11]. 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 participates 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 hydrolyze the same synthetic substrates cleaved by TOP and NEL, besides bioactive peptides like bradykinin and neurotensin [8]. Furthermore, 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 carboxypeptidase; 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.

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similar structural/functional activity between the mammalian and the bacterial oligopeptidase [8]. 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 position 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 mutagenesis in the enzyme catalytic activity and protein folding in order 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 resonance energy transfer (FRET) bradykinin-related peptides as substrates. 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. Materials and methods Protein model generation 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.expasy.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/software/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 another of OpdA in its ‘‘closed” state.

pET vector, containing cDNA encoding the desired protein (GE Healthcare, Chalfont St. Giles, UK), as previously described [27]. Recombinant proteins were purified to homogeneity by affinity chromatography 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 spectropolarimeter 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 dmol1). The spectrometer 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 incubation 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 The FRET peptides containing the fluorescent group ortho-aminobenzoic acid (Abz) and the acceptor N-[2,4-dinitrophenyl]ethylenediamine (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 Shimadzu, Tokyo, Japan) as described elsewhere [28]. The final deprotected 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 binary HPLC system from Shimadzu fitted with SPD-10AV Shimadzu UV–vis detector and Shimadzu RF-535 fluorescence detector. The system was coupled to an Ultrasphere C18 column (5 lm, 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 elution profile of the peptides was monitored by the absorbance at 220 nm and by the fluorescence emission at 420 nm following excitation 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 spectrometry (TofSpec-E, Micromass, Manchester, UK) and peptide sequencing with a PPSQ-23 protein sequencer (Shimadzu, Tokyo, Japan). Stock solutions of the peptides were prepared in DMSO, and the concentrations were measured spectrophotometrically using the molar extinction coefficient of 17,300 M1cm1 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 identified as OpdA Y607F and OpdA Y607A, here named Y607F and Y607A, respectively. The enzymes OpdA WT, Y607F and Y607A were expressed in E. coli DH5a using the pHis3 plasmid, a modified

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-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine), where X represents Ala, Gly, Leu, Phe, Tyr, Ser, Gln, Asn, Glu, Asp, His, Arg,

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Lys and Pro were tested. The kinetic parameters of peptide hydrolysis 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 converted 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 hydrolyzed. The inner-filter effect was corrected using an empirical equation [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 RF10AXL fluorescence detector coupled with an Ultrasphere C-18 column (5 lm, 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 nonlinear regression analysis using Grafit program [30].

K¼

Limit1 þ Limit2  10ðpHpK 1 Þ Limit2 þ Limit3  10ðpHpK 2 Þ 10ðpHpK 1 Þ þ 1

10ðpHpK 2 Þ þ 1

ð1Þ

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 constant, 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. Results and discussion Modeling studies The 3D structural model of TOP [20], NEL [19] and Dcp [26] revealed 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 also underwent significant distortion, 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 substrate 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-

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). 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 polyacrylamide 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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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 enzyme. Therefore, the site-directed mutagenesis of the Tyr607 residue did not promote significant changes in the secondary structure of the proteins. 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 different 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 mutants. 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

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 concentration 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 mutants 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 mM1 s1). Surprisingly, the peptides Abz–GHSPFRQ–EDDnp (kcat/Km = 1910 mM1 s1) and Abz–GKSPFRQ–EDDnp (kcat/Km = 1095 mM1 s1) that contain in P1 the positively charged residues His and Lys, respectively, were not as suitable for hydrolysis as Abz–GRSPFRQ–EDDnp. The hydrophobic aromatic residues Phe (kcat/Km = 1584 mM1 s1) and Tyr (kcat/Km = 2191 mM1 s1) were also well accepted by the S1 subsite. 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 mutants demonstrated that the presence the Tyr607 residue is essential 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 hydrolysis up to 2.1 lM enzyme concentration. This confirms the importance, not only of the hydroxyl group, but mainly of the aromatic ring on the 607th position for OpdA activity. Both mutant enzymes showed their highest kcat/Km values with peptides bearing aromatic amino acids on the P1 position, especially the Tyr residue.

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 related 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.

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R.Z. Lorenzon et al. / Archives of Biochemistry and Biophysics 500 (2010) 131–136 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

A G L F Y S Q N E D H R K P

OpdA WT

Y607F

Km (lM)

kcat (s

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

1

)

kcat/Km (mM s)

1

Y607A

Km (lM)

595 194 844 1584 2191 590 258 273 42 49 1910 10,358 1095 159

2.2 1.3 8.6 2.7 1.2 0.5 5 2.6

kcat (s

1

)

kcat/Km (mM s)

1

Km (lM)

0.014 18 0.008 6 (P–F) 0.2 2.1 0.1 36 0.065 55 0.004 7 0.05 10 0.02 7 kcat/Km = 0.25 (P–F)* 0.002 0.5 (P–F) 0.08 20 0.034 49 kcat/Km = 7 (X–S; P–F)* kcat/Km = 0.8 (P–F)*

4.7 4 0.7

kcat (s1)

kcat/Km (mM s)1

kcat/Km = 0.6 (X–S; P–F)* 0.02 0.3 (P–F) 0.032 4 (P–F) 0.025 4 0.064 10 0.003 0.3 (P–F) kcat/Km = 0.6 (X–S; P–F)* kcat/Km = 0.2 (X–S; P–F)* Resistant Resistant kcat/Km = 0.6* 0.005 3 kcat/Km = 0.5 (X–S; P–F)* Resistant

9.4 8.9 6.1 6.5 10

1.7

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.

kcat/Km mutant / k cat/Km WT

1

0.1

0.01

0.001

0.0001

A

G

L

F

Y

S

Q N Amino acid (X)

E

D

H

R

K

P

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. 4. NaCl influence in the hydrolysis of Abz–GFSPFRQ–EDDnp by OpdA WT (s),Y607F (d) and Y607A (h). In the ratio (k/k0), k0 is the kcat/Km value obtained in 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.

Fig. 5. pH dependence for hydrolysis of Abz–GFSPFRQ–EDDnp by OpdA WT (d), Y607F (s) and Y607A (j). The assays were performed as described in Material and methods section. Values are presented as kcat/Km.

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,

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the low catalytic efficiency shown in both mutants reflects the lack 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 recognition of the correct cleavage site shows that important chemical interaction performed by the Tyr607 residue are critical for catalytic activity. This is clearly shown by the results obtained with the mutants Y607F and Y607A which cannot perform their hydrolytic activity as well as the wild-type enzyme. Therefore, the low catalytic efficiency presented by both mutants reflects the importance of the contacts performed by the aromatic and hydroxyl groups of the tyrosine residue that are essential to anchor the substrate in the enzyme active site. Taken together, the present work shows the importance of Tyr607 in the catalytic efficiency of OpdA and the resemblance of this enzyme with the other members of the M3A subfamily. Similarly to what happens in other metallopeptidases as astacin and thermolysin [32,33], this specific Tyr residue might be crucial for substrate catalysis and could be relevant to stabilize the oxyanion intermediate formed after nucleophilic attack by water. Acknowledgments This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). References [1] [2] [3] [4] [5] [6] [7] [8]

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