Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase Vitor Oliveira 1 , Reynaldo Gatti 2 , Vanessa Rioli 3 , Emer S. Ferro 3 , Alberto Spisni 2,4 , Antonio C. M. Camargo 5 , Maria A. Juliano 1 and Luiz Juliano 1 1 Department of Biophysics, Escola Paulista de Medicina, Sa ˜ o Paulo, Brazil; 2 Centro de Biologia Molecular Estrutural, National Laboratory of Synchrotron Light (CBME-LNLS), Campinas, Brazil; 3 Department of Histology, Institute of Biomedical Sciences, Universidade de Sa ˜ o Paulo, Brazil; 4 Department of Experimental Medicine, University of Parma, Parma 43100, Italy; 5 Laboratory of Biochemistry and Biophysics, Instituto Butantan, Sa ˜ o Paulo, Brazil We report the recombinant neurolysin and thimet oligo- peptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 M for NaCl, KCl, NH 4 Cl and NaI, and 0.025–0.1 M for Na 2 SO 4 . Concentration higher than 0.2 N NH 4 Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na 2 SO 4 increased TOP activity. Neu- rolysin was strongly activated by NaCl, KCl and Na 2 SO 4 , while NH 4 Cl and NaI have very modest effect. High positive values of enthalpy (DH*) and entropy (DS*) of activation were found together with an unusual tempera- ture dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concen- tration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far- UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities. Keywords: protease; peptide; metalloprotease; fluorescence; enthalpy of activation in proteolysis; entropy of activation in proteolysis. Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zinc-dependent peptidases, members of the metallopeptidase M3 family and contain in their primary sequence the HEXXH motif [1,2]. Rat neurolysin has been the first member of the M3 family of which the 3D structure has been determined [3]. As it has been shown that this enzyme and thermolysin have common ancestors, the M3 family was thus included in the clan MA [4]. It is interesting to note that in the structure of neurolysin, the catalytic center is located in a deep channel [3], which limits the access only to short peptides [5,6]. This selectivity toward hydrolysis of oligopeptides was also verified for TOP [5–9]. The high primary sequence identity found for these related peptidases, which is about 65% [2], allows hypothesizing that they may share a similar folding including the deep channel that constitutes the neurolysin active site. This feature seems to prevent the unspecific cleavage of other proteins and it is of particular relevance for TOP and neurolysin as they are not expressed as inactive precursors and they are present in high amount as soluble enzymes in cytosol. On the other hand, membrane associ- ated form of TOP [10] and neurolysin have been identified [11,12]. The secretion of TOP has been reported in AtT20 [13,14] and MDCK cells [15] while neurolysin was showed to be secreted by astrocytes [16]. Efficient oligopeptidases are required to metabolize biologically active peptides before and after their interac- tion with cell receptors, this is particularly relevant with neuropeptides that lack classical reuptake mechanisms for recycling components into the cell. TOP exhibits charac- teristics of both metabolizing and processing enzymes, and has multiple peptide substrates as GnRH [17], neurotensin [18], bradykinin [19], somatostatin 1–14 [20], and nociceptin [21]. TOP also processes Met- and Correspondence to L. Juliano, Departamento de Biofisica, Escola Paulista de Medicina, Rua Treˆ s de Maio, 100, 04044-020 Sa ˜ oPaulo, SP, Brazil. Fax: + 55 11 5575 9040, Tel.: + 55 11 5575 9617, E-mail: juliano.biof@epm.br Abbreviations:Abz,ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine; IQF peptide, internally quenched fluorescent peptide, TOP, thimet oligopeptidase. Enzymes: thimet oligopeptidase (TOP, EC 3.4.24.15); neurolysin (EC 3.4.24.16). *Present address: Department of Experimental Medicine, University of Parma, Parma, 43100, Italy. (Received 10 April 2002, revised 3 July 2002, accepted 19 July 2002) Eur. J. Biochem. 269, 4326–4334 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03129.x Leu-enkephalin from the enkephalin-containing peptides [22], and the specific TOP inhibition increased Met-enke- phalin antinociception in rodents [23]. TOP and neurolysin are able to hydrolyze the biologically active peptide neurotensin (NT) in vitro and they could be participating in the catabolism of this biologically active peptide. In vivo experiments have been showed that NT degradation is blocked by TOP and neurolysin inhibitors [24] and a highly specific neurolysin inhibitor potentiated the neurotensin- induced antinociception of mice in the hot plate test when administrated intracerebroventricularly [25]. In addition, higher vertebrates produce large number of oligopeptides generated by the proteasome system due to proteolysis of intracellular and/or of foreign proteins. However, only some of these peptides are presented to the immune system on cell surface MHC class I molecules [26–28]. Recently it was reported the involvement of TOP in sorting and hydrolysis of the peptides generated by proteasomes [30–32]. A detailed analysis of the substrate specificity of neu- rolysin and TOP was reported using internally quenched fluorescent (IQF) peptides derived from bradykinin [5] and neurotensin [6]. An outstanding feature of the hydrolytic activities of neurolysin and TOP on these substrates is the variability of the cleavage sites, which is a consequence of modifications in size or in nature of the amino acids at different positions of the substrates [5,6]. The 3D structure determination of neurolysin supports this broad specificity turning up the possibility of a reorganization of the flexible loops of the enzyme binding site in order to accommodate the substrates [3]. This very particular mechanism of substrate interaction with a peptidase requires a detailed study of the milieu composition and temperature influence on neurolysin and TOP hydrolytic activities in order to understand their unusual variations of specificity [5,6] and broad spectrum of hydrolysis of biologically active peptides as described above. In the present work we report the neurolysin and TOP hydrolytic activities towards IQF peptides derived from Abz-GGFLRRVQ-EDDnp [Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine], in which Val was substituted by 11 different natural amino acids. This sequence was chosen because we have previously observed efficient hydrolysis by TOP of the peptide Abz- GGFLRRV-EDDnp at L-R bond and the addition of Gln at C-terminal site (Abz-GGFLRRVQ-EDDnp) resulted in two cleavages, at L–R and or at R–R bond. We made modifications at Val position in order to verify the influence of the nature of the amino acid at this position on determination of the cleavage sites and on the amount of their hydrolysis. This series of peptides was chosen because the P 2 ¢ and P 3 ¢ positions were demonstrated to be very determinant on the specificity of neurolysin and TOP [5]. The kinetic parameters of hydrolysis and the variations of the cleavage sites of this series of peptides by these two oligopeptidases were evaluated in different conditions of temperature and salts. MATERIALS AND METHODS Thimet oligopeptidase (TOP) The purified recombinant rat testes TOP (rTOP) was obtained as previously described [33]. Details about the procedures applied for enzyme characterization and active site titration were described elsewhere [5]. Neurolysin The recombinant cDNA of porcine liver neurolysin (cyto- solic form) was a kind gift from S. Hirose and A. Kato (Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan). Details concerning the expression system including plasmid constructions and vectors used were described elsewhere [34]. The procedures for expression and purification of recombinant porcine liver neurolysin were performed as previously reported for the recombinant rat testes TOP [33]. The methods for the determinations of enzyme purity and concentration were also previously described [6]. Peptide synthesis The IQF peptidescontaining N-[2,4-dinitrophenyl]-ethylene- diamine (EDDnp) attached to glutamine were synthesized by solid-phase strategy, which details are provided elsewhere [35]. An automated bench-top simultaneous multiple solid- phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the synthesis of all the peptides by the Fmoc- procedure. The final deprotected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 l, 22.5 · 250 mm) and a two-solvent system: (A) trifluoroacetic acid/H 2 O (1 : 1000) and (B) trifluoroacetic acid/acetonitrile/H 2 O (1 : 900 : 100). The column was eluted at a flow rate of 5 mLÆmin )1 with a 10 (or 30))50 (or 60)% gradient of solvent B over 30 or 45 min. Analytical HPLC was performed using a binary HPLC system from Shima- dzu with a SPD-10AV Shimadzu uv-vis detector and a Shimadzu RF-535 fluorescence detector, coupled to an Ultrasphere C-18 column (5l,4.6· 150 mm) which was eluted with solvent systems A and B at a flow rate of 1mLÆmin )1 and a 10–80% gradient of B over 20 min. The HPLC column eluates were monitored by their absorbance at 220 nm and by fluorescence emission at 420 nm follow- ing excitation at 320 nm. The molecular mass and purity of synthesized peptides were checked by MALDI-TOF mass spectrometry (TofSpec-E, Micromass) and/or peptide sequencing using a protein sequencer PPSQ-23 (Shimadzu Tokyo, Japan). Kinetic assays The Michaelis parameters were determined by initial rate measurements. The hydrolysis of the fluorogenic peptidyl substrates at 37 °Cin50m M Tris/HCl buffer pH 7.4 containing 100 m M NaCl was followed by measuring the fluorescence at k em ¼ 420 nm and k ex ¼ 320 nm in a Hitachi F-2000 spectrofluorometer. The 1-cm path-length cuvette containing 2 mL of the substrate solution was placed in a thermostatically controlled cell compartment for 5 min before the enzyme solution was added and the increase in fluorescence with time was continuously recor- ded for 5–10 min. For TOP an additional preincubation time of 5 min with 0.5 m M of dithiothreitol were applied before substrate addition. This amount of dithiothreitol was chosen because it provided the maximum enzyme activation in our conditions. The slope was converted into mols of Ó FEBS 2002 Modulation of thimet oligopeptidase and neurolysin activities (Eur. J. Biochem. 269) 4327 hydrolyzed substrate per minute based on the fluorescence curves of standard peptide solutions before and after total enzymatic hydrolysis. The concentration of the peptide solutions was obtained by colorimetric determination of the 2,4-dinitrophenyl group (17 300 M )1 Æcm )1 , extinction coef- ficient at 365 nm). The enzyme concentrations for initial rate determinations were chosen at a level intended to hydrolyze less than 5% of the substrate present in the reaction. The inner-filter effect was corrected using an empirical equation as previously described [36]. The kinetic parameters were calculated according Wilkinson [37] as well as by using Eadie–Hofstee plots. All the obtained data were fitted to nonlinear least square equations, using GRAFIT v3.0 from Erithacus Software [38]. The hydrolysis of the substrates cleaved at two peptide bonds by TOP and neurolysin can be represented as shown in Scheme 1, whose equation for velocity is Eqn (1). V t is the sum of the velocities of formation of the products (P a and P b ). V a max is kp a · [E] and V b max is kp b · [E], and [E] is the total enzyme concentration in the assay. All the obtained data with the peptides cleaved at two bonds fitted to nonlinear least square plot of Eqn (1). The overall V max was obtained from Eqn (1), whereas the separate values for V a max and V b max were calculated using the ratio of the areas taken from the integrated HPLC chromatogram analysis. Additional data and discussion about this kinetic interpret- ation can be found in more details in [5]. V t ¼ ½SÁðV a max þ V b max Þ K s þ½S ð1Þ For the specificity rate constants (k cat /K m ) which were determined under first-order conditions, we used substrates concentrations 10-fold less than K m . The obtained first- order rate constants were divided by the total enzyme concentration to provide k cat /K m . As the products Abz- GGFL, Abz-GGFLR and their respective C-terminal fragments were resistant to hydrolysis by TOP, and the products Abz-GGFLR, Abz-GGFLRR and their respect- ive C-terminal fragments were also resistant to hydrolysis by neurolysin, we could determine the specificity rate constants (k cat /K m ) under first-order conditions, even for the peptides hydrolyzed at two peptide bonds. This procedure was used in the assays conduced at different temperatures and at different salt concentrations [5]. Temperature dependence of the hydrolysis reaction rates of the substrates by neurolysisn and TOP The temperature dependence of the rate constants was determined in thermostated cell holders. The reactions were started after the thermal equilibrium had been reached in the cell. Typically the reactions were carried out in 1 mL of 50 m M Tris/HCl buffer pH 7.4 containing 100 m M NaCl. Activation parameters were calculated from the linear plots of ln(k/T)vs.1/T (Eqn 2), where k is the rate constant, R is the gas constant (8.314 JÆmol )1 Æ K )1 ), T is the absolute temperature, N A is Avogadro’s number, h is Planck’s constant, the enthalpy of activa- tion DH* ¼ –(slope)8.314 JÆmol )1 , the entropy of activa- tion DS* ¼ (intercept – 23.76)8.314 JÆmol )1 ÆK )1 .Thefree energy of activation DG*, was calculated from Eqn (3) (T ¼ 298.15 K). ln k T  ¼ ln R N A h  þ DS Ã R À DH Ã RT ð2Þ DG Ã ¼ DH Ã À TDS Ã ð3Þ Dependence of the hydrolysis reaction rates by neurolysisn and TOP on concentration and chemical nature of salts The dependence of the rate constants according to the concentration and the chemical nature of salts were determined in 1 mL of 50 m M Tris/HCl buffer pH 7.4 containing different concentrations of NaCl, KCl, NH 4 Cl (0–2 N), NaI and Na 2 SO 4 (0–1 N). A strong fluorescence quenching caused by the I – ion did not permitted the experiments with 2 N NaI. Determination of cleaved bonds The cleaved bonds were identified by isolation of the fragments by HPLC either comparing the retention times of the products fragments with synthetic peptides encompas- sing the expected hydrolysis products and/or by molecular mass. The molecular masses were determined by MALDI- TOF mass spectrometry and/or by sequencing, using a protein sequencer PPSQ-23 (Shimadzu Tokyo, Japan). Amino-acid analysis The amino-acid compositions, the concentration of the peptides and the purified rTOP were determined as follows: the samples were digested for 22 h at 110 °Cin6NHCl containing 1% phenol in vacuum sealed tubes and then subjected to amino-acid analysis using a pico-Tag station [39]. Circular dichroism CD spectra were recorded at Jasco J-810 spectropolarimeter with a Peltier system of cell temperature control. The system was routinely calibrated with an aqueous solution of recrystalized d-10 camphorsulphonic acid. Ellipticity is reported as mean residue molar ellipticity, [h] (degÆcm 2 Ædmol )1 ). The spectrometer conditions were typi- cally: spectral range 195–260 nm, 100 mdeg sensibility; 0.2 nm resolution; 4 s response time; 20 nmÆmin )1 scan rate, 7 accumulations at the appropriate temperature (10, 25 or 37 °C). The 100 mdeg sensibility is used in our routine that leads to the lower noise-signal relationship. The control baseline was obtained with solvent and all the components without the proteins. All the data were obtained with three Scheme 1. 4328 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002 different solutions of the proteins. The quality of data was certified by the correspondence of the amount of secondary structures obtained by CD data deconvolution with those from the 3D structure of neurolysin. The errors of prediction on the range 195–260 nm and 200–260 nm were 5% using the CDNN program [40]. RESULTS Kinetic parameters for the hydrolysis of IQF peptide series Abz-GGFLRRXQ-EDDnp by TOP and neurolysin Table 1 shows the kinetic parameters of the hydrolysis by TOP and neurolysin of the substrates on the series Abz- GGFLRRXQ-EDDnp and their peptide bonds cleaved in the kinetic measurement conditions. TOP hydrolyzed the peptides I to VI only at R–R bonds, which contain at position X basic or aromatic amino acids, besides Pro and Ala. On the other hand, the peptides VII to XII, which contain hydrophobic or acidic amino acids at the X position, besides Asn, were hydrolyzed either at R–R or at L–R bonds, but preferentially at the R–R bond, except the peptide XII that contains Asp. The higher specificity constant (k cat /K m ) values were obtained with the substrates cleaved only at R–R bond, and the catalytic constant (k cat ) was the predominant component. Peptide XII and XIII (Qf 7 in Table 1) were exceptions in terms of preferential cleavage site by TOP, which is directed to L–R bond by Asp in peptide XII or by the absence of Gln in the peptide XIII, however, their k cat /K m values were the lowest in the series. On the other hand, the highest k cat /K m values obtained with TOP were for substrates I and II containing Arg and His at the X position, respectively. Neurolysin, like TOP, hydrolyzed all the substrates at R–R bond, but the alternative cleavage site was at R–X bond in peptides II to IV, VII, IX and X, which contain at the X position of the series Abz-GGFLRRXQ-EDDnp essentially hydrophobic amino acids. The peptides hydro- lyzed exclusively at R–R bonds contain at the X position charged amino acids (Arg, Asp and Glu) or amino acids with small hydrophobic side chain (Ala, Pro and Val). The highest k cat /K m value for neurolysin was observed with the hydrolysis of the substrate with Ala (peptide VI), and the lowest k cat /K m values was the peptide with Asp (peptide XII) at the X position. Temperature dependence of the substrate hydrolysis by TOP and neurolysin The preference of cleavage at the L–R or R–R bond for TOP and at the R–R or R–X bond for neurolysin in the case of the substrates containing Val, Asp and Ile at the X position in the studied series were determined at temperature range 10–37 °C. These peptides were chosen due to their different preferences for hydrolysis of the susceptible bonds. Both peptidases were stable at 37 °C for more than 20 min, which was longer than that used in all enzyme assays. At 45 °C significant decrease of activity was observed in the first 5 min, possibly due to enzyme denaturation. A significant increase in the percentage of hydrolysis by TOP at the R–R bond was observed by decreasing temperature with the peptides containing Val or Asp. No significant changes were observed with the peptide having Ile or with any other substrate assayed with neurolysin (Table 2). Both, the dithiothreitol used for activation of TOP [41] and pH variations from 6 to 9 did not affect the ratio of hydrolysis between the two hydrolytic sites by the two oligopeptidases. The k cat /K m values were determined, at the temperature range 10–37 °C in the presence of 0.1 M NaCl, for hydrolysis of Qf 7 and the substrates of the series Table 1. Kinetics parameters for hydrolysis by TOP and neurolysin of the peptides derived from Abz-GGFLRRXQ-EDDnp. The parameters were calculated as mean value ±S.D., which was never greater than 7%. The kinetic parameters for the hydrolysis of the substrates with two cleavage sites were obtained using Eqn 2. k cat /K m ¼ (k a cat + k b cat )/K m . L–R, R–R and R–X indicate the cleavage sites. Qf 7 is the abbreviation used for the peptide Abz-GGFLRRV-EDDnp. The kinetic experiments were conduced at 37 °Cin50m M Tris/HCl buffer containing 0.1 M NaCl. For XIII, cleavage site is L–R. Number X TOP (24.15) Neurolysin (24.16) k cat (s )1 ) K m (l M ) k cat /K m (l M )1 Æs )1 ) k cat (s )1 ) K m (l M ) k cat /K m (l M )1 Æs )1 ) L–R R–R R–R R–X I R – 11 1.5 7.3 3.3 – 2.3 1.4 II H – 10 1.4 7.1 1.6 0.6 1.7 1.3 III Y – 19 4.3 4.4 0.3 0.2 0.6 0.8 IV F – 15 3.6 4.2 0.2 0.3 0.7 0.7 V P – 7.4 2.2 3.4 1.8 – 1.2 1.5 VI A – 15 4.8 3.1 3.2 – 0.5 6.4 VII N 1.6 5.5 1.8 3.9 0.6 0.3 1.5 0.6 VIII V 1.7 2.8 1.7 2.6 1.4 – 2.1 0.7 IX I 0.8 4.3 2.0 2.5 0.7 0.1 1.8 0.4 X L 1.8 3.5 2.5 2.1 0.2 0.5 0.7 1.0 XI E 0.7 2.2 1.5 1.9 1.1 – 1.9 0.6 XII D 3.4 1.6 3.2 1.6 1.0 – 3.3 0.3 XIII Qf 7 0.7 – 1.7 0.4 2.0 – 2.2 0.9 Ó FEBS 2002 Modulation of thimet oligopeptidase and neurolysin activities (Eur. J. Biochem. 269) 4329 Abz-GGFLRRXQ-EDDnp containing Val, Ile and Ala at the X position. The peptide containing Asp were only assayed with TOP. Linear Eyring plots (ln[k/T ]vs.1/T ) were obtained for the hydrolysis of Qf 7 by both enzymes and for the hydrolysis of the substrate containing Val by neurolysin. The Eyring plots for the hydrolysis by TOP of the peptides containing Ile, Val and Asp deviated from the linearity above 25 °C. The plot for the reaction of the peptide containing Ala with TOP was not linear in all studied range of temperature (Fig. 1A). The Eyring plots obtained for neurolysin reactions with the peptides containing Ala and Ile at the X position gave two linear fittings, above and below % 22 °C (Fig. 1B), indicating different rate-limiting steps at each temperature range (above and below 22 °C). The DG* DH*andDS* values were taken from Eyring plots and are shown in Table 3. In addition to the temperature dependence of the catalytic constants accord- ing to the substrates, the positive and high values of DH* and DS* for hydrolysis are of note. Influence of the NaCl on TOP and neurolysin activities The influence of NaCl concentration on k cat /K m values of TOP and neurolysin activities on the peptides containing Ala, Val, Asp and Ile at the X position in the series Abz- GGFLRRXQ-EDDnp was examined in the salt concen- tration range 0–0.5 M , and the data are presented in Table 4. The k cat /K m values increased with the increasing of NaCl concentration for all the assayed substrates, except with the peptide containing Asp. The higher NaCl effects were observed for the hydrolysis of the peptide with Ile at the X position by neurolysin and TOP (Table 4). NaCl was observed also to modulate the preference of both enzymes for their cleavage sites, namely, the increase of NaCl concentration further enhanced the percentage hydrolysis by TOP and neuro- lysin at R–R bond (Table 4). The activation parameters of TOP and neurolysin activities on Qf7 were also determined in the presence of 2 M NaCl. In this condition, the Eyring plots obtained for TOP and neurolysin gave two linear fittings, above and below % 22 °C, which contrast with linear plots obtained in the absence of salt. Similar to all others, substrates with similar temperature behavior resulted in DH*andDS* values at temperature range 25–37 °C significantly lower than those at 10–20 °C(Table3). Influence of the chemical nature of salts on the TOP and neurolysin activities Using Qf7 as a reference substrate, which was hydrolyzed only at L–R bond, we studied the effects of different salts on TOP and neurolysin activities. The results are shown in Fig. 2A,B, respectively. TOP was activated by all the assayed salts (NaCl, KCl, Na 2 SO 4 ,NH 4 Cl and NaI) at low concentrations. However, the increase of NH 4 Cl or NaI concentrations reduced TOP activity, in contrast to NaCl, KCl and Na 2 SO 4 that progressively increased the enzyme activity from 0.5 till 2 N salt concentrations. In the case of neurolysin, NaCl, KCl and Na 2 SO 4 exhibited the more intense activation effect (one order of magnitude more than that with TOP), and the effects of salts were proportional to their concentration. NH 4 Cl and NaI exhibited small activation without any inhibitory activity as observed with TOP. Fig. 1. Eyring plots for substrate hydrolysis reaction by TOP and neu- rolysin. (A) Eyring plots for the hydrolysis carried out with TOP on Qf 7 (black circles), Abz-GGFLRRAQ-EDDnp (open circles) and Abz-GGFLRRVQ-EDDnp (black squares). (B) Eyring plots for the hydrolysis carried out with neurolysin on Qf 7 (black circles), Abz- GGFLRRAQ-EDDnp (open circles). The hydrolysis reactions were carried out in Tris buffer 50 m M , pH 7.4 containing NaCl 100 m M . Table 2. Influence of temperature on the preference cleavage sites of TOP on the peptides derived from Abz-GGFLRRXQ-EDDnp. The kinetic experiments were conduced in 50 m M Tris/HCl buffer con- taining 0.1 M NaCl. T °C Cleaved bond %, TOP X ¼ V X ¼ D X ¼ I L–R R–R L–R R–R L–R R–R 10 18 82 39 61 11 89 20 26 74 50 50 9 91 30 27 73 59 41 11 89 37 38 62 64 36 17 83 4330 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Circular dichroism spectra of TOP and neurolysin The CD spectra of TOP and neurolysin show a predomi- nance of a helical structures as shown in Fig. 3 (without smoothing and curve fitting). For neurolysin spectra, the results obtained in the deconvolution of the CD data, using the CDNN program [40], are consistent with the helix content found in the neurolysin crystal structure, as the helix content from the crystal structure was 53% [3]; and from the deconvolution of CD at 37 °C in the absence of NaCl (195– 260 nm) was 51%. For the spectrum of TOP in the same conditions the deconvolution indicated 45% of a helix content. This result is close to that of neurolysin and consistent with consensus secondary structure prediction obtained from different algorithms ( DPM , DSC , GOR 4, HNNC , PHD , PREDATOR , SIMPA 96, POPM ) performed at the internet site http://npsa-pbil.ibcp.fr. The variation of the NaCl concentration from 0 to 1 M did not affect the a helical component in a detectable manner in the far-UV CD assay. Changes in the secondary structure of TOP and neurolysin with the temperature (10 and 37 °C) were also not detected in the CD experiments in the presence and in the absence of NaCl. DISCUSSION TOP and neurolysin hydrolyzed all the assayed substrates of the series Abz-GGFLRRXQ-EDDnp at three or four amino acids from their C-terminal ends. These results agree with previously reported hydrolysis by both enzymes of IQF peptides derived from neurotensin [6] and the hydrolysis of repetitive sequences of tri-peptides [42] by TOP three or four amino-acid residues from the C-terminal end of the substrates. TOP and neurolysin also hydrolyze IQF peptides derived from bradykinin by a similar way. In this case, depending on the sequence and size of the substrates, the hydrolysis were observed 6–10 amino acids from the C-terminal end of the peptides but with very low efficien- cies [5]. Comparatively, neurolysin hydrolyzed closer to C-terminal end than TOP the series Abz-GGFLRRXQ- EDDnp, as also observed for hydrolysis of neurotensin derivatives [6]. In fact, despite the R–R bond being the Table 3. Activation parameters for TOP and neurolysin reactions with the substrates of the series Abz-GGFLRRXQ-EDDnp and Abz-GGFLRRV- EDDnp (Qf 7). The kinetic experiments were conduced in 50 m M Tris/HCl buffer containing 0.1 M NaCl. The parameters were calculated as mean value ± S.D. Enzyme Substrate Temperature Range a °C DG* kJÆmol )1b DH* kJÆmol )1 DS* JÆmol )1 ÆK )1 TOP Qf 7 10–37 43.4 134 ± 3 304 ± 6 Qf7 c 10–20 40.9 203 ± 5 544 ± 10 25–37 40.7 109 ± 3 229 ± 5 X=V 10–25 40.0 159 ± 2 399 ± 5 X=D 10–25 41.4 141 ± 14 334 ± 31 X=I 10–25 40.2 152 ± 1 375 ± 2 Neurolysin Qf7 10–37 44.2 139 ± 1 318 ± 3 Qf7 c 10–20 31.5 177 ± 4 488 ± 10 25–37 32.8 68 ± 2 118 ± 4 X=V 10–37 43.3 94 ± 4 170 ± 7 X=I 10–20 43.4 202 ± 7 532 ± 18 25–37 44.5 97 ± 4 176 ± 6 X=A 10–20 36.9 138 ± 1 339 ± 1 25–37 36.8 80 ± 3 145 ± 4 a Temperature range where the Eyring plots are linear. b At 298.15 K (25 °C). c Parameters determined in the presence of 2 M of NaCl. Table 4. Influence of NaCl concentration on the specificity constant (k cat /K m ) for hydrolysis of peptides derived from Abz-GGFLRRXQ-EDDnp by TOP and neurolysin. The unit of k cat /K m is l M )1 Æs )1 . The effect of NaCl on the variation of cleavage sites for neurolysin was perfomed only for the substrate containing Ile. The kinetic experiments were conduced at 37 °Cin50m M Tris/HCl pH 7.4. NaCl ( M ) X=A k cat /K m X=V a k cat /K m L–R R–R X=D a k cat /K m L–R R–R X=I a k cat /K m L–R R–R R–I TOP 0 2.6 1.4 46 54 2.8 76 24 2.1 22 78 0.1 3.4 1.8 37 63 1.7 64 36 2.7 15 85 0.5 8.0 3.7 20 80 0.9 33 67 6.1 9 91 Neurolysin 0 6.9 0.6 0.4 0.5 68 32 0.1 7.6 0.9 0.3 0.5 83 17 0.5 11 1.5 0.3 1.0 93 7 a Substrates with two cleavage sites, at L–R and R–R (or R–I in neurolysin) bonds, which amount of each cleavage is presented in percentage (%). Ó FEBS 2002 Modulation of thimet oligopeptidase and neurolysin activities (Eur. J. Biochem. 269) 4331 preferred cleavage site for both enzymes in the series Abz- GGFLRRXQ-EDDnp, TOP also hydrolyzed the L–R bond while neurolysin hydrolyzed the R–X bond. These observations are in accordance with the hydrolysis by recombinant neurolysin and TOP of natural substrates, such as bradykinin, neurotensin, metorphinamide, dynor- phin A 1–8 and angiotensin I [34]. The 3D structure of rat neurolysin [3] demonstrated that the substrate binding site is a channel, which amino-acid chains of its wall are connected by flexible loops and open coil regions. As a consequence, it is tempting to speculate that these flexible structures in neurolysin and TOP can accommodate peptides inside their substrate binding chan- nels with different degree of restrictions, which could be responsible for the absence of specificity, particularly for S 1 subsite. The displacement of the cleavage site to R–R bond on the hydrolysis of the substrates Abz-GGFLRRXQ- EDDnp at low temperatures and at high NaCl concentra- tions (Tables 2 and 4) suggested modifications on the channel binding site of neurolysin and TOP, better accom- modating the R–R residues for hydrolysis. In addition, the kinetic parameters k cat /K m varied with NaCl concentration, and the extent of it was dependent on the substrate (Table 4) and on the nature of the salts (Fig. 2). Therefore, the structures of TOP and neurolysin could be changed by salts, as a similar manner as the recently described activation of recombinant prostate-specific antigen (PSA) by Hofmei- ster salts [43]. The activation of PSA by high salt concen- tration was interpreted as a result of the salt interaction over the surface of the protein, and one possible way to reduce the unfavourable interaction is to reduce the protein surface by conformational change to a more compact structure, that resulted in a more active enzyme. However, in the cases of TOP and neurolysin, this effect on their activities should be localized in limited regions because the CD spectra collected in the absence of NaCl and in the presence of 1 M NaCl at three different temperatures, did not indicate Fig. 2. Influence of different salts on the k cat /K m of the hydrolysis of Qf 7 by TOP (A) and neurolysin (B). In the relation (k/k 0 ), k 0 is the k cat /K m value obtained in 50 m M Tris, pH 7.4 in the absence of any salt and k is the k cat /K m value obtained in a determined salt concentration. The concentration is presented in normality (N). Fig. 3. CD spectra of TOP and neurolysin collected at 37 °CinTris 50 m M pH 7.4 in the absence (full circles) or in the presence of 1 M NaCl (open triangles). These data are without curve fitting or smoothing. 4332 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002 detectable changes in their secondary structures. The intrinsic mechanism of activation of TOP and neurolysin by salts was not determined, however, change of rate- limiting step or the speed up of isomerization of the enzyme- substrate complex were described for other peptidases [44,45]. At low salt concentrations the predominant effects seems to be due to the shielding of charges present in the enzymes and in the substrates, as suggested the results obtained with the peptide containing X ¼ Asp (Table 4). Finally the activation of TOP and neurolysin by increasing concentration of NaCl was also verified with the substrate Abz-GFSPFIQ-EDDnp, which does not have charged side chains (results not shown) indicating that NaCl affects TOP and neurolysin structures. It is noteworthy the unusual temperature dependence of the k cat /K m with different substrates as showed by the Eyring-plots for TOP and neurolysin reactions (Table 3 and Fig. 1). With the substrate Qf 7 (with 0.1 M NaCl) the variation of the k cat /K m values with the temperature fits Eqn (2), giving linear Eyring-plots with both TOP and neurolysin. A similar linear Eyring plot was also obtained for the reaction of neurolysin with the substrate containing Val. On the other hand, deviations from the linearity were observed in the experiments with TOP above 25 °C (Fig. 1), and breaks in the plots were verified in the neurolysin experiments at 22 °C. Regarding TOP, this deviation from linearity is not due to enzyme instability at 37 °C, because we have checked this with specific experiments, and, in addition, in the temperature range 45 °Cto55°C, the enthalpy of inactivation of TOP in the presence of 0.1 M NaCl was 250 kJÆmol )1 , which is significantly higher than the enthalpy of activation of the reactions, as shown in Table 3. Deviation from linearity of Eyring plot may arise from changes in the rate limiting step [46] and/or from alterations in the enzyme structure with the temperature. In the temperature experiments with neurolysin and TOP, where a break in the Eyring plots occurs showing two linear sections can be interpreted as two different rate limiting steps at each temperature range [46]. Similar unusual temperature dependence of the k cat /K m ratio according with the substrate was observed for the oligopeptidase B [47]. The activation parameters were estimated for the tem- perature ranges in which linear Eyring plots were obtained (Table 3). In all reactions for both TOP and neurolysin markedly positive enthalpies and entropies of activation were verified. Positive entropy of activation can be associ- ated with reorganization of the protein structure, which may involve an unfolding process or changes in the water layer around the reactants [46]. On the other hand, as the substrates must go inside a channel to find the catalytic machinery, a considerable amount of water should be lost from the substrates, and in this case the entropy contribu- tion will be positive. This interpretation could be relevant considering the activities of neurolysin and TOP on neuropeptides containing free or amidated C-terminal carboxyl group, as more water should be organized around to the free C-terminal carboxyl group and the hydrolysis of these substrates should be more sensitive to changes of temperature and ionic strength. Finally, differences in the specificities and effects of salts and temperature between the two enzymes were significant, although not large, and could be related to limited differences in the primary structure widespread in the sequences of both enzymes. ACKNOWLEDGEMENTS This work was supported by the Fundac¸ a ˜ odeAmparoa ` Pesquisa do Estado de Sa ˜ o Paulo (FAPESP), Conselho Nacional de Desenvolvi- mento Cientı ´ fico e Tecnolo ´ gico (CNPq), and Human Frontiers Science Program (RG 00043/2000-M) and Ministry of Innovation, University and Research (MIUR), Italy. 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