RESUMO
Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two-dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.
Assuntos
Peptídeos Cíclicos/química , Pseudomonas/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Hidrogênio/química , Lactonas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Software , Trifluoretanol/farmacologiaRESUMO
Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Peptídeos Cíclicos/toxicidade , Pseudomonas/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Lipídeos de Membrana/química , Membranas Artificiais , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Pseudomonas/química , Pseudomonas/genética , Coelhos , Esteróis/química , Propriedades de SuperfícieRESUMO
Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.
Assuntos
Toxinas Bacterianas/toxicidade , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Lipídeos de Membrana/química , Peptídeos Cíclicos/toxicidade , Pseudomonas/patogenicidade , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Condutividade Elétrica , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Pseudomonas/química , Pseudomonas/genéticaRESUMO
Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.
Assuntos
Simulação por Computador , Peptídeos Cíclicos/química , Pseudomonas/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica , SoluçõesRESUMO
The structure of the corpeptins, bioactive lipodepsipeptides produced in culture by Pseudomonas corrugata, the causal agent of tomato pith necrosis, has been determined. The combined use of FAB-mass spectrometry, NMR spectroscopy and chemical procedures has allowed us to assign the following primary structure to the peptide moiety: Dhb-Pro-Ala-Ala-Ala-Val-Val-Dhb-Hse-Val-alle-Dhp-Ala-Ala-Ala-Val-D hb-aThr-Ala-Dab-Ser-Ile with the terminal carboxy group closing a macrocyclic ring on the hydroxy group of the allo-threonine residue. The N-terminus is in turn acylated by 3-hydroxydecanoate in corpeptin A and by cis-3-hydroxy-5-dodecenoate in corpeptin B. Some preliminary data on the biological activity of corpeptins are included.
Assuntos
Peptídeos Cíclicos/química , Pseudomonas/fisiologia , Acilação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Ácidos Decanoicos/análise , Hidrólise , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Similarly to other Pseudomonas lipodepsinonapeptides, pseudomycin A inhibits proton extrusion from maize roots, promotes closure of stomata in Vicia faba, necrosis of tobacco leaves, haemolysis of human erythrocytes, affects H(+)-ATPase activity and proton translocation in plasma membrane vesicles, and stimulates succinate respiration in pea mitochondria. In general, the biological activities of pseudomycin A are lower than those of syringomycin-E, the prototype member of this family of bacterial metabolities. This difference might depend on the diverse number and distribution of charged residues in the peptide moiety of these compounds.
Assuntos
Proteínas de Bactérias/farmacologia , Peptídeos Cíclicos/farmacologia , Plantas/efeitos dos fármacos , Pseudomonas/química , Sequência de Aminoácidos , Proteínas de Bactérias/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Pisum sativum/efeitos dos fármacos , Pisum sativum/fisiologia , Peptídeos Cíclicos/toxicidade , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Tóxicas , ATPases Translocadoras de Prótons/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/metabolismo , Zea mays/efeitos dos fármacos , Zea mays/metabolismoRESUMO
The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.
Assuntos
Toxinas Bacterianas/química , Peptídeos Cíclicos/química , Pseudomonas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/farmacologia , Fungos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Doenças das Plantas/microbiologia , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Relação Estrutura-AtividadeRESUMO
Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The conformation of syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-2-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three-dimensional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported.
Assuntos
Toxinas Bacterianas/química , Peptídeos Cíclicos/química , Conformação Proteica , Pseudomonas/química , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Ácidos Graxos/química , Hidrólise , Lipoproteínas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The Pseudomonas syringae pv. syringae phytotoxins syringomycin-E and syringopeptins 22-A and 25-A reversibly and noncompetitively inhibit purified H(+)-ATPase solubilized from plasma membrane of maize roots. Moreover, they increase the passive permeability to protons in phosphatidylcholine/phosphatidylethanolamine liposomes. Both effects are more pronounced with syringopeptins than with syringomycin-E. Activity on phospholipid bilayers is detectable at phytotoxin concentrations not affecting H(+)-ATPase activity.
Assuntos
Toxinas Bacterianas/farmacologia , Lipossomos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Pseudomonas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Solubilidade , Zea mays/citologia , Zea mays/enzimologiaRESUMO
A saprophytic fluorescent bacterium (strain M1) isolated from wheat was identified as Pseudomonas syringae and shown to produce the cyclic lipodepsipeptides, syringomycin E and syringopeptin SP25A. M1 grew in planta but did not affect germination or cause disease symptoms in wheat. The findings show that the production of these metabolites, generally regarded as plant virulence factors, does not correlate with plant pathogenicity.
Assuntos
Toxinas Bacterianas/metabolismo , Peptídeos Cíclicos/biossíntese , Pseudomonas/metabolismo , Triticum/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Divisão Celular/fisiologia , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Peptídeos Cíclicos/metabolismo , Triticum/citologiaRESUMO
Several authors' changes should have been included in the response by D. B. Wheeler et al. to the technical comment "Identification of calcium channels that control neurosecretion" (4 Nov., p. 830). A sentence reading, "Comparisons with experiments using higher concentrations of carrier protein (1.0 mg/mL) revealed no significant differences in the rate of efficacy of omega-Aga-IVA action" should have been inserted before the last sentence in the legend of figure 1. The measure "30 nM" (not 20 nM) should have appeared 19 lines from the bottom of the last column on page 830. The second-to-last sentence in that paragraph should read, "Increasing the duration of exposure to 30 nM omega-Aga-IVA from 20 min (1) to 45 min revealed significant inhibition of synaptic transmission (n = 9)." Reference "(15)" (not 16) should have appeared seven lines from the top of the first column on page 831.
Assuntos
Proteínas de Bactérias/química , Pseudomonas/metabolismo , Treonina/análogos & derivados , Ácido Aspártico/análogos & derivados , Proteínas de Bactérias/biossíntese , Biossíntese Peptídica , Peptídeos/química , Treonina/análiseRESUMO
Fusicoccin affects several physiological processes regulated by the plasma membrane H(+)-ATPase in higher plants while other organisms having P-type H(+)-ATPases (e.g., fungi) are fusicoccin-insensitive. We have previously shown that fusicoccin binding to its receptor is necessary for H(+)-ATPase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the H(+)-ATPase from maize. In this paper we show that fusicoccin sensitivity can be conferred on the H(+)-ATPase from Neurospora crassa, a fungus insensitive to fusicoccin. In fact, H+ pumping by purified H(+)-ATPase from Neurospora crassa reconstituted into liposomes containing crude or partially purified fusicoccin receptors from maize was markedly enhanced by fusicoccin. The stimulation of H+ pumping by fusicoccin is dependent upon pH, fusicoccin, and protein concentration, as was reported for the system reconstituted with both proteins from maize.
Assuntos
Glicosídeos/farmacologia , Proteínas de Plantas , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Neurospora crassa/enzimologia , Proteolipídeos , Transdução de Sinais , Zea maysRESUMO
The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. This is in turn N-acylated by 3,4-dihydroxytetradecanoate in pseudomycin A, by 3-hydroxytetradecanoate in pseudomycin B, by 3,4-dihydroxyhexadecanoate in pseudomycin C, and by 3-hydroxyhexadecanoate in pseudomycin C'. Some preliminary data on the biological activity of pseudomycin A are reported.
Assuntos
Antifúngicos/química , Proteínas de Bactérias/química , Peptídeos/química , Pseudomonas/química , Sequência de Aminoácidos , Aminoácidos/análise , Antifúngicos/farmacologia , Proteínas de Bactérias/farmacologia , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/análise , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/farmacologia , Plantas/efeitos dos fármacos , Plantas/metabolismoRESUMO
Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor.
Assuntos
Proteínas de Plantas , Receptores de Superfície Celular/isolamento & purificação , Zea mays/química , Sítios de Ligação , Membrana Celular/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Glicosídeos/metabolismo , Ensaio Radioligante , Receptores de Superfície Celular/metabolismoRESUMO
Biochemical properties of fusicoccin receptors are strongly influenced by the phospholipid environment. In this report we have studied the effect of different exogenous phospholipases on fusicoccin binding ability of both plasma membrane and solubilised receptors. Among the phospholipases tested only phospholipase A2 showed an inhibitory effect on fusicoccin binding. In particular, the influence of this enzyme on the time course and reversibility of the fusicoccin binding reaction was studied. The inhibitory effect of phospholipase A2 was the consequence of fatty acid release. The usual fatty acids of plasma membrane phospholipids were active in inhibiting the interaction of fusicoccin with its receptors. It is concluded that a phospholipid associated to the fusicoccin receptor might play a significant role in the modulation of binding.
Assuntos
Glicosídeos/metabolismo , Fosfolipases A/metabolismo , Proteínas de Plantas , Receptores de Superfície Celular/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Lipídeos/farmacologia , Fosfolipases A2 , Sementes/metabolismo , Zea mays/metabolismoRESUMO
The spectroscopic properties of syringomycin E, an antibiotic lipodepsinonapeptide associated with pathological states in plants, have been investigated by uv absorbance and CD spectroscopies, and by the synthesis of relevant model compounds. Initial studies [E. Vaillo, A. Ballio, P. L. Luisi, and R. M. Thomas (1990) in Peptides 1990, Giralt, E. & Andreu, D., Eds., Escom Scientific, Leiden, Netherlands] suggested that a significant contribution to the spectra was due to the presence of a zdehydroaminobutyric acid residue in the amino acid sequence. The model peptides N-Boc-L-Phe-delta zAbu-OMe and its analogue, N-Boc-L-Phe-L-Thr-OMe, lacking the unsaturated bond, were synthesized using standard solution chemistry, and a detailed investigation was made in which the spectra of the models and that of syringotoxin (an antibiotic closely related to syringomycin E but without a Phe residue) were compared with those of syringomycin E under a variety of solvent conditions. The uv absorbance spectra of both N-Boc-L-Phe-delta zAbu-OMe and syringomycin E clearly showed the presence of the unsaturated residue while the CD spectra were complex, environmentally sensitive, and contained contributions from both the delta zAbu and Phe residues. In the course of these studies extinction coefficients were obtained for syringomycin E and its dipeptide model. The origins of the uv and CD spectra are discussed in detail, and a comparison is made with the spectra of other, similar lipopeptide antibiotics. Finally, a structural model for syringomycin is proposed in which the changes induced in the spectrum by alterations in the solvent environment are accommodated.
Assuntos
Proteínas de Bactérias/química , Sequência de Aminoácidos , Antibacterianos/química , Toxinas Bacterianas/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Modelos Químicos , Dados de Sequência Molecular , Espectrofotometria UltravioletaRESUMO
Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H(+)-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V(max) of the enzyme. Fusicoccin stimulation of maize H(+)-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).
RESUMO
The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudomonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin-producing strains afforded 3-hydroxydecanoyl-Dhb-Pro-Val-Val-Ala-Ala-Val-Val-Dhb-Ala-Val-Ala- Ala-Dhb-aThr-Ser-Ala-Dhb-Ala-Dab-Dab-Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin-producing strain and a syringotoxin-producing strain synthesize 3-hydroxydecanoyl-Dhb-Pro-Val-Ala-Ala-Val-Leu-Ala-Ala-Dhb-Val-Dhb- Ala-Val-Ala-Ala-Dhb-aThr-Ser-Ala-Val-Ala-Dab-Dab-Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Peptídeos/química , Pseudomonas/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , FotoquímicaRESUMO
The binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10 degrees C to 40 degrees C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu-proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] greater than human leucocyte elastase congruent to human cathepsin G congruent to subtilisin Carlsberg congruent to bovine alpha-chymotrypsin greater than bovine alpha-chymotrypsinogen A congruent to porcine pancreatic elastase congruent to bovine beta-trypsin. The serine (pro)enzyme-inhibitor complex formation is an entropy-driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift of the invariant hystidyl catalytic residue from approximately to 6.9 in the free serine proteinases and bovine alpha-chymotrypsinogen A to congruent to 5.1 in the serine (pro)enzyme-inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine (pro)enzyme-inhibitor contact regions.