Eur. J. Biochem. 268, 1206±1212 (2001) q FEBS 2001
S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids Lira C. Alves1, Robson L. Melo1, Sanya J. Sanderson2, Jeremy C. Mottram3, Graham H. Coombs2, Giuseppe Caliendo4, Vincenzo Santagada4, Luiz Juliano1 and Maria A. Juliano1 1
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de SaÄo Paulo, Brazil; 2Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, UK; 3Wellcome Centre for Molecular Parasitology, University of Glasgow, UK; 4Dipartimento di Chimica Farmaceutica e Tossicologica, UniversitaÁ di Napoli, Italy.
We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8DCTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115±122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoylFRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf ), 4-guanidine-phenylalanine (Gnf ), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf ), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-aminobenzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of
Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (kcat /Km 12 000 mm21´s21) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (kcat /Km 27 000 mm21´s21) were the best substrates for CPB2.8DCTE. In contrast, ortho-aminobenzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]ethylenediamine were very resistant and inhibited this enzyme with Ki values of 23 nm and 30 nm, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a Ki of 40 nm. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8DCTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8DCTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P1 position.
Protozoa of the genus Leishmania contain an abundance of cysteine proteinases (CPs) of the papain family. They exhibit stage-regulated expression and the CP activity of
Leishmania mexicana is considerably greater in the mammalian amastigote form than in the promastigote forms that live in the sandfly vector [1]. This observation led to the discovery that one class of CPs exists as multiple isoenzymes [2±6], which are encoded by a tandem array of 19 similar CPB genes [6±10]. This class of CPs, together with homologues from other leishmanias [11,12] and other trypanosomatids such as Trypanosoma cruzi (cruzipain or cruzain) [13,14] and Trypanosoma brucei [15] are cathepsin L-like and are characterized by the presence of an unusual 100-amino-acid C-terminal extension, which in some cases is highly glycosylated. The trypanosomatid CPs have been shown to be attractive targets for novel antiparasite agents [16±18] and a recombinant CP isoenzyme of L. mexicana (designated CPB2.8DCTE) was expressed, without its C-terminal extension, in Escherichia coli and purified, for detailed analysis [19]. In mammals, the major CPs are the lysosomal cathepsins B and L and they are implicated in many physiological processes [20±23]. The kinetic properties of these different CPs have been studied using a variety of substrates and
Correspondence to M. A. Juliano, Rua Tres de Maio, 100, 04044-020 SaÄo Paulo, Brazil. Fax: 1 55 11 5575 9040, Tel.: 1 55 11 5575 9617, E-mail:
[email protected] Abbreviations: Abz, ortho-amino-benzoyl; Aca, 4-aminocyclohexylalanine; Ama, 4-aminomethyl-cyclohexyl-alanine; Cha, cyclohexyl alanine; EDDnp, N-[2,4-dinitrophenyl]-ethylenediamine; CP, cysteine proteinase; CPB2.8DCTE, recombinant Leishmania mexicana cysteine proteinase CPB2.8 lacking the C-terminal extension; Amf, 4-aminomethyl-phenylalanine; Gnf, 4-guanidine-phenylalanine; Iaf, 4-aminomethyl-N-isopropyl-phenylalanine; Ppa, 4-piperidinyl-alanine; Pya, 3-pyridyl-alanine. Enzymes: cathepsin L (EC 3.4.22.15); cathepsin B (EC 3.4.22.1); Leishmania mexicana cysteine proteinase (no EC number); papain (EC 3.4.22.2). (Received 16 October 2000, revised 7 December 2000, accepted 14 December 2000)
Keywords: cathepsin L; cruzain; cysteine proteinase of Leishmania mexicana; papain.
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Substrate specificity of cysteine proteinases (Eur. J. Biochem. 268) 1207
inhibitors. It is well established that the primary determinant of specificity for papain and cathepsins B and L is the S2 subsite [24,25]. Hydrophobic residues are preferred at the P2 position of substrates for papain and cathepsin L, but cathepsin B also accepts basic residues there. This difference is due to the presence of a glutamic acid residue at S2 of cathepsin B [26,27]. The S1 subsites of papain and cathepsin L are not as selective as the S2, accepting a wide range of residues. However, a preference of the papain S1 subsite for basic residues has been shown. This is related to interactions of the substrate Arg guanidine group side chain with papain Asp158 [28]. The analysis of the specificity of CPB2.8DCTE [29] revealed that it has strict preferences for the S1 to S3 subsites, accepting substrates containing basic amino acids in P1, hydrophobic residues in P2 and Lys in P3. In contrast, the specificity of the primed subsites was shown to be broad. Moreover, it was demonstrated that peptides containing hydrophobic amino acids at the P1 position, with basic and hydrophobic amino acids at P3 and P2, respectively, were resistant to hydrolysis by CPB2.8DCTE but nevertheless had affinities in the nanomolar range [30]. The substrate specificity of cruzain, the major cysteine proteinase in T. cruzi, was investigated using a portion-mixing combinatorial library (one bead±one peptide, [31]) and intramolecularly quenched fluorescence substrates derived from cystatin peptides [32]. It was observed that both hydrophobic and basic amino acids are accepted at the P2 position and the enzyme has a broad specificity for the positions P3, P1 and P 0 2. These substrate preferences of papain and cruzain were recently confirmed using combinatorial fluorogenic libraries and a recombinant form of cruzain without the C-terminal extension [33]. In our program aimed at developing efficient inhibitors of CPs of Leishmania and T. cruzi, it was relevant to investigate substrates for CPB2.8DCTE containing at the P1-position amino acids that combine both a bulky hydrophobic side chain and a basic group. The rationale was that such residues would take advantage of both the high hydrolytic susceptibility of substrates with basic amino acids at P1 and the high affinity of peptides with hydrophobic residues at this position. We synthesized one series of internally quenched fluorogenic peptides derived from the sequence AbzFRSRQ-EDDnp, (where Abz represents ortho-amino-benzoyl and EDDnp represents N-[2,4-dinitrophenyl]-ethylenediamine) which is based on the human kininogen sequence at the C-terminal region of bradykinin, and replaced Arg with non-natural, basic amino acids. These amino acids were designed to combine a large hydrophobic and/or aromatic group with a positively charged group at their side chains. The following amino acids were synthesized and introduced in these peptides: 4-aminomethyl-phenylalanine (Amf ), 4-guanidine-phenylalanine (Gnf ), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf ), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca) (see Fig. 1 for the structures of side chains). The kinetic parameters for CPB2.8DCTE and cruzain were determined, as were the substrate cleavage sites. For comparison, the series derived from AbzFRSRQ-EDDnp were also assayed with human cathepsin L and papain.
M AT E R I A L S A N D M E T H O D S Synthesis of protected non-natural basic amino acids All the amino acids were characterized by 1H NMR (Bruker AMX-500) and mass spectroscopy (LCQ Thermoquest-Ion Trap) and the data were consistent with the considered structures. L-4-aminomethyl-phenylalanine
(Amf )
Fmoc-l-Phe(4-CH2NH-Boc) was prepared according to the procedure, appropriately modified, detailed in Stokker et al. [34]. The key step of the Amf synthesis, introduction of the 4-[N-(trichloroacetyl)-amino]methyl group is accomplished readily via acid-catalyzed, nuclear amidoalkylation of l-Phe and is followed by acid hydrolysis to yield Amf dihydrochloride in modest yield (< 30%) but in a high state of chiral purity. Treatment with di-tert-butyldicarbamate in butylalcohol afforded the 4-[N-(benzyloxycarbonyl)amino]methyl-l-phenylalanine. The Fmoc-group was introduced by Fmoc-N-hydroxysuccinamide ester. L-4-Aminomethyl-N-isopropyl-phenylalanine
(Iaf )
Fmoc-l-Phe[4-CH2NH-iPr-Boc] was obtained starting from Fmoc-l-Phe(4-CH2NH2) by reductive alkylation with acetone. Treatment with di-tert-butyl dicarbonate in tertbutyl alcohol afforded the final product in good yield. L-Trans-4-aminomethyl-cyclohexyl-alanine
(Ama)
Fmoc-l-trans-cyclohexylalanine (Cha) (4-CH2NH-Boc) was prepared starting from Fmoc-l-Phe(4-CH2NH-Boc) according to a previously described procedure [35]. Catalytic hydrogenation of the starting compound in the presence of
Fig. 1. Side chain structure of the basic non-natural amino acids employed to substitute Arg2 in the peptide Abz-F1-R2-S3-R4-QEDDnp.
1208 L. C. Alves et al. (Eur. J. Biochem. 268)
platinum oxide gave a mixture of Fmoc-l-cis/trans-Cha(4CH2NH-Boc). The trans isomers were separated by preparative liquid chromatography. L-Cis/trans-4-amino-cyclohexyl-alanine
(Aca)
Fmoc-l-cis/trans-Cha-(4-NH-Boc) was prepared starting from Fmoc-4-nitro-l-phenylalanine as previously described [35]. Catalytic hydrogenation of the nitro derivative in the presence of platinum oxide gave a mixture of Fmoc-cisand trans-aminocyclohexyl-l-alanine. Treatment with ditert-butyldicarbonate in tert-butyl alcohol afforded the final product in good yield. L-4-Guanidine-phenylalanine(Gnf )
Fmoc-l-phenylalanine [4-guanidino(Boc)2] was prepared according to a previously reported procedure, which was appropriately modified [36], starting from p-nitro-phenylalanine which, after its amine group has been protected by a Fmoc-group, was reduced so as to convert the NO2 group into an -NH2 group. The resulting derivative was reacted with a guanidination agent, such as 1-guanidino-3,5dimethylpyrazole nitrate, to obtain the Fmoc-Gnf derivative. The guanidine moiety was successively protected as Boc-by di-tert-butyl dicarbonate in tert-butyl alcohol (yield 60%). L-3-Pyridyl-alanine
(Pya)
Fmoc-l-(3-Pyr)-Ala was prepared as previously described [37]. Treatment of 3-bromopyridine with methyl 2-acetamidoacrylate in the presence of Tris (dibenzyl deneacetone) dipalladium under an inert atmosphere gives good yield of prochiral enamide. The enamide is hydrogenated in the presence of chiral Rh catalyst to give the l-methyl-2acetamido-3-(3-pyridyl)propanate. The free amino acid was released by hydrolysis and treatment with Fmoc-Nhydroxysuccinamide ester afforded the final compound in good yield. L-4-Piperidinyl-alanine
(Ppa)
Fmoc-l-Ppa(Boc)-OH was prepared according to a previous reported procedure [38,39]. The title compound was prepared in nine steps from 3-(4-pyridyl)-acrylate acid using Evans chiral auxiliary. Peptide synthesis All the intramolecularly quenched fluorogenic peptides contained EDDnp attached to glutamine. This is a necessary result of the solid-phase peptide synthesis strategy employed, the details of which are provided elsewhere [40]. An automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the solid-phase 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 mm, 22.5 250 mm) and a two-solvent system: (a) trifluoroacetic acid/H2O (1 : 1000) and (b) trifluoroacetic acid/acetonitrile/H2O (1 : 900 : 100). The column was eluted at a flow rate of 5 mL´min21 with a 10 (or 30)250% (or 60%) gradient of
q FEBS 2001
solvent B over 30 or 45 min. Analytical HPLC was performed using a binary HPLC system from Shimadzu with a SPD-10AV Shimadzu UV-vis detector and a Shimadzu RF-535 fluorescence detector, coupled to an Ultrasphere C-18 column (5 mm, 4.6 150 mm) which was eluted with solvent systems A1 (H3PO4 /H2O, 1 : 1000) and B1 (acetonitrile/H2O/H3PO4, 900 : 100 : 1) at a flow rate of 1.7 mL´min21 and a 10±80% gradient of B1 over 15 min. The HPLC column eluates were monitored by their absorbance at 220 nm and by fluorescence emission at 420 nm following excitation at 320 nm. The purity of obtained peptides were checked by amino-acid sequencing, performed with a Shimadzu Sequencer model PPSQ-23 and by MALDI-TOF in the reflectron mode (TofSpec-E from Micromass Manchester, UK). Enzymes CPB2.8DCTE was expressed, purified and activated as previously described [19]. The concentration of the enzyme stock solution (11.4 mm) was determined by active-site titration with human cystatin C, which was a generous gift from M. Abrahamson (University of Lund, Sweden), using Z-Phe-Arg-NH-Mec as the substrate. Human cathepsin L and papain were obtained according to Carmona et al. and MeÂnard et al. [41,42], respectively. Cruzain was obtained as previously described [43]. The molar concentrations of the enzyme solutions were determined by active site titration with E-64 [trans-epoxysuccinyl-l-leucylamido-(4guanidino)butene] according to Barrett and Kirschke [44]. Enzymatic hydrolysis of fluorescent quenched substrates Hydrolysis of the fluorogenic peptide substrates by CPB2.8DCTE and human cathepsin L was carried out in 0.1 m sodium acetate, 2.0 mm EDTA, 200 mm NaCl, pH 5.5. Cruzain was assayed in 0.1 m sodium phosphate, 400 mm NaCl, 2.0 mm EDTA, pH 6.3 and papain in 0.1 m sodium phosphate, 1.0 mm EDTA, pH 6.8. All kinetic analyses were done at 37 8C with 15-min enzyme preincubation in 10 mm dithiothreitol. The Abz-peptidyl-QEDDnp substrate hydrolysis were monitored by measuring the fluorescence at lem 420 nm with excitation at lex 320 nm using a Hitachi F-2000 spectrofluorometer, as previously described [45,46]. The standard hydrolysis conditions were strictly maintained for different substrates. The enzyme concentration was varied from 0.006 nm, for the best substrates, to 12 nm for the less susceptible ones. The kinetic parameters were calculated as previously described [47]. The standard deviations of Km and kcat determinations were in no case higher than 6% of the obtained value. When these conditions could not be met because the peptide was resistant to hydrolysis, or was hydrolyzed but at a low rate (less than 1.5 nmol´min21) at the upper limit of enzyme concentration, or if the substrate inhibited the enzyme at concentrations lower than the estimated Km value, the Ki was then determined for competitive inhibition assays of the peptides, which were obtained according to Nicklin and Barrett [48]. Determination of the cleavage point in the assayed substrates The bonds cleaved were identified by isolation of the fragments by HPLC and the retention times of the fluorescent
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Substrate specificity of cysteine proteinases (Eur. J. Biochem. 268) 1209
Table 1. Kinetic constants for hydrolysis of the substrates Abz-FXSRQ±EDDnp by L. mexicana CPB2.8DCTE, cruzain, human cathepsin L and papain. Variations of the peptides at position X corresponds to the P1. Conditions were 0.1 m sodium acetate pH 5.5 (CPB2.8DCTE and cathepsin L), 0.1 m Sodium phosphate pH 6.3 (cruzain), pH 6.8 (papain), 2.0 mm EDTA, 200 mm NaCl, 10.0 mm dithiothreitol preactivation for 15 min, 37 8C. Enzyme range concentrations were 0.1±14.0 nm (cathepsin L), 0.05±2.0 nm (papain), 2.0±6.5 nm (CPB2.8DCTE), 0.1±0.3 nm (cruzain). Z-Phe-Arg-NH-Mec was used as substrate for inhibition studies. The standard error for the determinations of Km and kcat were lower than 6%. CPB2.8DCTE
No.
X
1 2 3 4 5 6 7 8 9 10
R H F Amf Gnf Iaf Pya Ama Ppa Aca
Km (mm)
kcat (s21)
0.2 1.5
1.1 3.0 Ki 1.2 0.2 2.7 0.7 Ki Ki Ki
0.1 0.5 0.1 0.5
Cruzain kcat /Km (mm21´s21) 5500 2000 65.0 nm 12 000 400 27 000 1400 23.0 nm 44.0 nm 30.0 nm
Cathepsin L
Km (mm)
kcat (s21)
0.4 1.0 0.2 0.1
2.6 6500 3.6 3600 1.9 9500 2.9 29 000 Ki 113.0 nm Ki 52.0 nm 1.8 9000 Ki 40.0 nm 1.9 19 000 3.2 16 000
0.2 0.1 0.2
kcat /Km (mm21´s21)
Abz-containing fragments compared with authentic synthetic sequences and/or by amino-acid sequencing.
R E S U LT S Hydrolysis of peptides derived from Abz-F1-R2-S3-R4-Q-EDDnp Table 1 shows the kinetic parameters for the hydrolysis of the peptides derived from Abz-F1-R2-S3-R4-Q-EDDnp (reference substrate, peptide 1) in which Arg2 was substituted by His, Phe, and by non-natural basic amino acids, the structures of which are shown in Fig. 1. All the peptides were hydrolyzed only at the X2-S3 bond (X all substitutions of Arg2) by CPB2.8DCTE, cruzain, and cathepsin L, therefore with these peptides we have information about the S1 specificity of these enzymes. On the other hand, papain hydrolyzed the peptides at X2-S3 bond but also the S3-R4 bond to approximately the same extent in the substrate where Arg2 was substituted by Phe (peptide 3), and preferentially at X2-S3 bond (approximately 80%) and only less at the S3-R4 bond in the substrates with Amf, Gnf, Iaf and Pya (peptides 4±7). Therefore the kinetic parameters for these cleavages are apparent values as they represent the hydrolysis of two peptide bonds. The reference substrate (peptide 1) was hydrolyzed by CPB2.8DCTE, cathepsin L and papain with higher kcat /Km values compared with the substrate with Phe (peptide 3). Cruzain preferred Phe at P1 in comparison to Arg. The substrate with His (peptide 2), of which the imidazole group is positively charged at pH 5±6, was well hydrolyzed by all the enzymes, although with less efficiency than reference substrate (peptide 1). Curiously, the presence of Phe at P1 in peptide 3 resulted in slow hydrolysis by CPB2.8DCTE and at concentrations higher than 50 nm substrate inhibition was observed. The Ki value for this peptide was determined in competitive conditions, in order to evaluate its affinity for CPB2.8DCTE, which
Papain
Km (mm)
kcat (s21)
0.2 0.3 0.3 0.1 0.2 0.1 0.3
2.7 13 500 2.2 7333 1.3 4333 1.8 18 000 1.1 5500 3.6 36 000 0.4 1333 Ki 54.0 nm Ki 56.0 nm 1.6 16 000
0.1
kcat /Km (mm21´s21)
Km (mm)
kcat (s21)
kcat /Km (mm21´s21)
0.42 0.23 0.32 0.21 0.25 0.12 0.33 0.36 2.03 0.26
5.4 1.6 2.1 0.7 1.1 1.8 1.6 4.8 3.6 3.0
12 857 6956 5562 3333 4400 15 000 4848 13 333 1773 11 538
was significantly high (Ki 65 nm). The substrates with the basic non-natural amino acids with an aromatic side chain Amf, Gnf, Iaf and Pya (peptides 4±7) were hydrolyzed by CPB2.8DCTE and the peptides with Amf and Iaf (4 and 6) were the best substrates for this enzyme. The peptides containing Ama, Ppa and Aca (peptides 8± 10), in which the aromaticity was absent, presented the same behavior with CPB2.8DCTE as the substrate with Phe (peptide 3). However, they inhibited the enzyme more efficiently, and peptide 8 with the amino acid Ama presented the lowest Ki value. Cruzain hydrolyzed quite efficiently the substrates with Amf, Ppa and Aca (peptides 4, 9 and 10). In contrast, the peptides with Gnf, Iaf and Ama were resistant to hydrolysis and competitively inhibited the enzyme, particularly the peptide 8 with the amino acid Ama. The substrates with Phe and Pya (peptides 3 and 7) were hydrolyzed with very similar kinetic parameters by cruzain, indicating that the enzyme accommodates at S1 the heterocyclic structure of pyridyl group similarly to the phenyl group of Phe. This contrasts with CPB2.8DCTE that presented high affinity to the substrate with a phenyl group, whereas peptides with pyridyl were more susceptible but with lower affinity. The substrates were hydrolyzed by human cathepsin L with kinetic parameters very similar to those obtained with CPB2.8DCTE, except the peptides with Phe and Aca (3 and 10) that were efficiently hydrolyzed by cathepsin L. Papain hydrolyzed very efficiently most of the substrates, and the higher kcat values were observed with the basic aliphatic side chains as in Arg, Ama, Ppa and Aca (peptides 1 and 8±10).
DISCUSSION The three-dimensional structures of CPs have shown that the P1 side chain makes few interactions as it extends straight out of the cleft towards the solvent (reviewed in [49]). However, the irreversible inhibitors that were
1210 L. C. Alves et al. (Eur. J. Biochem. 268)
cocrystallized with cruzain had at P1 amino acids such as Ala with a short side chain [50,51]. The definition of substrate-binding sites of papain-like CPs was revised and analysis of the crystal structures has now shown that the substrate residues P2, P1 and P1 0 bind at well-defined binding sites within the active site cleft [52]. Cathepsin B was crystallized with an inhibitor containing a benzyl group bound to the hydroxyl group of Ser, and in this case the benzyl group (Bzl) was interpreted to occupy S1 0 pocket [26]. Recently we have obtained kinetic parameters for hydrolysis by cathepsin B of substrates containing at P1 Cys(Bzl) and Ser(OBzl) and these data support this structural interpretation [53]. In addition, a systematic study of the substrate specificity of papain using the combination of substrate library methodology and molecular dynamics calculations showed that the high acceptance of Arg at the S1 pocket of papain is due to the interaction of the substrate Arg with the carboxylate of papain Asp158 [28]. The results for papain presented in this paper confirm this preference of the enzyme for substrates with basic residues at P1. Moreover, the four best substrates contained at this position Arg, Iaf, Ama and Aca, indicating that the substrate susceptibility is dependent on the structure of the basic amino acid at P1. However, the greatest dependence of enzyme±substrate interaction on the nature and structure of the amino acid at the P1 position was observed with CPB2.8DCTE. This enzyme was able to hydrolyze substrates containing at P1 Arg, His or amino acids that combine aromatic basic side chains such as Amf, Gnf, Iaf or Pya. The peptides with Phe or amino acids that combine aliphatic basic side chains such as Ama, Ppa and Aca had high affinity but were very resistant to hydrolysis, the Ki values for them being in the range 23±65 nm. These results indicate that CPB2.8DCTE requires for a productive hydrolysis very selective interactions that depend on the fitting of the P1 side chain to the S1 pocket. A speculative explanation for the resistance to hydrolysis by CPB2.8DCTE of the peptides with Phe, Ama, Ppa and Aca is the possibility that the side chains of these amino acids occupy the S1 0 subsite as described for cathepsin B [26,53], which would give a high affinity to the peptides. According to this hypothesis, we observed (data not shown) that Phe at P1 0 position in the series of peptides derived from Abz-KLRFSKQ-EDDnp are hydrolyzed by CPB2.8DCTE with Km values around 50 nm, indicating a high affinity of S1 0 subsite for hydrophobic amino acids. Details of this binding will be gained only with the resolution of the three-dimensional structure of the enzyme, which is in progress in our laboratory. Although with the substrates used in this work we observed a good parallel between the kinetic data of CPB2.8DCTE and cathepsin L, the Aca was resistant to the Leishmania enzyme and well hydrolyzed by cathepsin L (Table 1, peptide 10). Unfortunately we were not able to separate the two forms of Aca (cis/trans), but clearly the selectivity of a single isomer is likely to be higher than we detected here. Cruzain preferentially hydrolyzes substrates with aromatic or aliphatic groups at P1 but with a small basic substituting group (compare in Fig. 1 the structures of Amf with Gnf and Iaf, or Ama with Ppa and Aca). Although the reported three-dimensional structure described that the methyl group of Ala at the P1 position of the inhibitor sticks out to the solvent [51], other interactions probably occur with amino acids that have larger side chains.
q FEBS 2001
In conclusion, we have demonstrated that S1 of CPB2.8DCTE has more restricted specificity than the other CPs tested and the data also suggest that it may be possible to design efficient inhibitors with amino acids such as Ama or Aca at the P1 position. Cruzain is less restrictive for P1 and in contrast to CPB2.8DCTE, the results from using the various substrates indicate that it is not reasonable to expect to find a good inhibitor for cruzain by manipulating the substrates at P1 position.
ACKNOWLEDGEMENTS This work was supported by the INCO-DC program (EU Contract Number ERBIC18CT970225), the Brazilian Research Foundations: FAPESP and PADCT. We acknowledge and thank for assistance: in Brazil, I.Y. Hirata for peptide sequencing, E. Galucci de Andrade for peptide synthesis and purification, and M.H. Cezari Sedenho for the expression and purification of recombinant cruzain and human cathepsin L; in the UK, K. Pollock, for work on production of the recombinant Leishmania enzyme. J.C.M. is a MRC Senior Research Fellow.
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