RESUMEN
We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Fasciola hepatica/enzimología , Calicreínas/metabolismo , Amidas , Secuencia de Aminoácidos , Animales , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Hidrólisis , Quininógeno de Alto Peso Molecular/metabolismo , Cininas/metabolismo , Datos de Secuencia Molecular , Coloración y Etiquetado , Especificidad por Sustrato , ortoaminobenzoatosRESUMEN
We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
Asunto(s)
Catepsina B/metabolismo , Catepsinas/metabolismo , Endopeptidasas , Colorantes Fluorescentes/metabolismo , Fragmentos de Péptidos/síntesis química , Catepsina L , Cisteína Endopeptidasas , Factor X/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Calicreínas/metabolismo , Hígado/enzimología , Papaína/metabolismo , Especificidad por Sustrato , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures epsilonNH2-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (orthoaminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBz1), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH2Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S'(1) subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in substrates containing Cys(SBzl) amino acid at P1 seems to compensate the absence of adequate S2-P2 interaction in the hydrolysis of the peptides having Pro or Ala at P2 position.
Asunto(s)
Aminoácidos/metabolismo , Catepsina B/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes/metabolismo , Humanos , Hidrólisis , Cinética , Papaína/metabolismo , Péptidos/síntesis química , Conformación Proteica , Especificidad por SustratoRESUMEN
Little is known about the species specificity of tissue kallikrein-kininogen interaction since the kinetic parameters for Lys-bradykinin release from kininogen by tissue kallikreins from different animal species have not been reported. We have now determined the kinetic parameters for hydrolysis by human and porcine tissue kallikrein, hK1 and pK1, respectively (Berg et al., 1992) of two series of intramolecularly quenched fluorogenic peptides having the sequences that flank the scissile Arg-Ser or Met-Lys bond in human and bovine kininogen. Results have shown that peptides having sequences from human kininogen are better substrates for hK1 and peptides derived from bovine kininogen are better substrates for pK1. Kinetic data for hydrolysis of the Arg-Ser bond showed that differences in the interaction of residue(s) in positions P2'-P10' contribute to the efficiency of the cleavage and may be responsible for differences in their susceptibilities to the two kallikreins. Significant variations in the kinetic data were observed for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension at sites P3-P9. The highest k(cat)/Km value in the hydrolysis of Abz-[Gln370-Gln381]-bkng-EDDnp by pk1 demonstrates an important interaction of subsites S5-S4 with Gln and Thr residues in the bovine kininogen segment. A Gln370-Gln391 bovine kininogen fragment used to study the cleavage of both Met-Lys and Arg-Ser bonds in the same molecule confirmed the importance of an extended interaction site for species specificity among tissue kallikreins.
Asunto(s)
Calicreínas de Tejido/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Bovinos , Colorantes Fluorescentes/metabolismo , Humanos , Hidrólisis , Lisina/metabolismo , Metionina/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Serina/metabolismo , Especificidad de la Especie , Especificidad por Sustrato , PorcinosRESUMEN
OBJECTIVE: Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). DESIGN: We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids). METHODS: Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods. RESULTS: Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE. The best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively. This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril. CONCLUSIONS: We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group. They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum.
Asunto(s)
Peptidil-Dipeptidasa A/análisis , Espectrometría de Fluorescencia/métodos , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Captopril/farmacología , Cloruros/farmacología , Ácido Edético/farmacología , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Péptidos/metabolismo , Peptidil-Dipeptidasa A/sangre , Especificidad por SustratoRESUMEN
We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Galbeta)Ser or (Glcalpha,beta)Tyr at P2 or P3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8-His-Leu-Val-Ile-His-Asn14 of human angiotensinogen, in which [GlcNAcbeta]Asn was introduced before Phe8 and/or after His13 and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal3)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glcalpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P'4 glycosylated peptide [Abz-F-H-L-V-I-H-(GIcNAcbeta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K(m) and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P'4 and P4 glycosylated substrates.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Angiotensinógeno/química , Angiotensinógeno/metabolismo , Animales , Catepsina D/metabolismo , Bovinos , Glicosilación , Humanos , Calicreínas/antagonistas & inhibidores , Calicreínas/metabolismo , Papaína/antagonistas & inhibidores , Papaína/metabolismo , Pepsina A/metabolismo , Fragmentos de Péptidos/síntesis química , Ratas , Renina/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismo , Calicreínas de Tejido , Tripsina/metabolismoRESUMEN
The major isoform of Trypanosoma cruzi cysteinyl proteinase (cruzipain) has generated Lys-bradykinin (Lys-BK or kallidin), a proinflammatory peptide, by proteolysis of kininogen. The releasing of this peptide was demonstrated by mass spectrometry, radioimmunoassay, and ileum contractile responses. The kinin-releasing activity was immunoabsorbed selectively by monoclonal antibodies to the characteristic COOH-terminal domain of cruzipain. To determine the hydrolysis steps that account for the kininogenase activity of cruzipain, we synthesized a fluorogenic peptide (o-aminobenzoyl-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-S er-Pro-Phe-Arg389-Ser390-Ser-Arg-Ile-NH2) based on the sequence Leu373 to Ile393 of the human high molecular weight kininogen. The hydrolysis products from this peptide were isolated by high performance liquid chromatography, and Lys-BK was characterized as the major released kinin by mass spectrometry. Intramolecularly quenched fluorogenic peptides spanning the Met379-Lys380 and Arg389-Ser390 bradykinin-flanking sequences were then used to assess the substrate specificity requirements of the parasite-derived protease compared with two COOH-terminal truncated recombinant isoforms (cruzain and cruzipain 2). In contrast to the high catalytic efficiency of parasite-derived cruzipain, the recombinant proteinases cleaved the bradykinin-flanking sites at markedly different rates. In addition, we also demonstrated that cruzipain activates plasmatic prekallikrein, which would be a second and indirect way of the parasite protease to release bradykinin.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Calicreínas/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/metabolismo , Activación Enzimática , Glicoproteínas/metabolismo , Humanos , Hidrólisis , Calidina/metabolismo , Cinética , Quininógenos/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P'4 to P'11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P'11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.