RESUMEN
Glutamic acid derived hydroxamates were identified as potent and selective inhibitors of procollagen C-proteinase, an essential enzyme for the processing of procollagens to fibrillar collagens. Such compounds have potential therapeutic application in the treatment of fibrosis.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Glutamatos/síntesis química , Glutamatos/farmacología , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Metaloendopeptidasas/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Proteína Morfogenética Ósea 1 , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Cinética , Inhibidores de la Metaloproteinasa de la Matriz , Relación Estructura-ActividadRESUMEN
The parallel synthesis of novel inhibitors of procollagen C-terminal proteinase is described. The synthetic strategy allowed for the facile synthesis of a large number of side-chain diversified diamino acid hydroxamates, of which the D-diaminopropionic acid derivatives were shown to be single digit nanomolar PCP inhibitors.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Proteína Morfogenética Ósea 1 , Indicadores y Reactivos , Cinética , Relación Estructura-ActividadRESUMEN
A discussion of the solid-phase synthesis of ornithine derived sulfonamide hydroxamic acids is illustrated. These analogues are shown to be potent, non-peptide inhibitors of procollagen C-proteinase (PCP).
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Ácidos Hidroxámicos/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Ornitina/síntesis química , Sulfonamidas/síntesis química , Aminoácidos/química , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Metaloendopeptidasas/metabolismo , Ornitina/análogos & derivados , Ornitina/química , Ornitina/farmacología , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
A solid-phase approach to the rapid synthesis of di- and tripeptide-like hydroxamic acids is presented. These compounds are shown to be potent inhibitors of procollagen C-proteinase (PCP).
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Ácidos Hidroxámicos/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteína Morfogenética Ósea 1 , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacologíaRESUMEN
OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno/metabolismo , Colagenasas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular , Inhibidores de la Metaloproteinasa de la Matriz , Osteoartritis/metabolismo , Adulto , Agrecanos , Técnicas de Cultivo , Femenino , Humanos , Lectinas Tipo C , Masculino , Metaloproteinasa 13 de la Matriz , Persona de Mediana Edad , Desnaturalización Proteica , Proteoglicanos/efectos de los fármacos , Proteoglicanos/metabolismoRESUMEN
Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.
Asunto(s)
Isoenzimas/metabolismo , Peroxidasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Etanol/metabolismo , Humanos , Cinética , Masculino , Proteínas de la Membrana , Modelos Químicos , Vesículas Seminales/enzimología , Ovinos , Espectrofotometría AtómicaRESUMEN
A plot of the pH dependence of kcat/KM for human stromelysin-1 (HS) exhibits a narrow range of maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.5-8.5. In contrast, the pH profiles that have been reported for other members of the matrix metalloproteinase (MMP) family are bell-shaped and exhibit neutral pH optima. We hypothesized that the anomalous pH dependence of HS reflects the ionization of His-224, a residue located in a flexible loop that contributes to the S1' binding pocket of the enzyme. HS is the only known MMP that has a histidine in this position. To test this hypothesis, the H224Q mutant of the short form (lacking the C-terminal hemopexin-like domain) of HS (sHS) has been prepared and studied. The pH profile of H224Q sHS is bell-shaped and similar to those reported for other MMPs. Although H224Q and wild-type sHS possess similar activities at pH <6, the kcat/KM of H224Q sHS is more than 5-fold greater than that of the wild-type enzyme at pH >7. These data strongly suggest that the deprotonation of His-224 attenuates the activity of HS, thereby accounting for its low pH optimum and the characteristic shoulder in its pH profile. This attenuation of activity appears to be predominantly a KM effect, reflecting a decrease in the affinity of the enzyme for the peptide substrate.
Asunto(s)
Histidina/fisiología , Metaloproteinasa 3 de la Matriz/fisiología , Glutamina/genética , Hemopexina/química , Histidina/química , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Metaloproteinasa 3 de la Matriz/química , Metaloproteinasa 3 de la Matriz/genética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Conformación Proteica , Especificidad por SustratoRESUMEN
Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages. Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family. To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described. In this report, we have examined the action of purified recombinant human ME (rHME), produced in Escherichia coli, on elastin and other extracellular matrix proteins. On a molar basis, rHME is approximately 30% as active as human leukocyte elastase in solubilizing elastin. rHME also efficiently degrades alpha1-antitrypsin (alpha1-AT), the primary physiological inhibitor of human leukocyte elastase. In addition, rHME efficiently degrades fibronectin, laminin, entactin, type IV collagen, chondroitan sulfate, and heparan sulfate. These results suggest that HME may be required for macrophages to penetrate basement membranes and remodel injured tissue during inflammation. Moreover, abnormal expression of HME may contribute to destructive processes such as pulmonary emphysema and vascular aneurysm formation. To further understand the specificity of HME, the initial cleavage sites in alpha1-AT have been determined. In addition, the hydrolysis of a series of synthetic peptides with different P'1 residues has been determined. rHME can accept large and small amino acids at the P'1 site, but has a preference for leucine.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Macrófagos/enzimología , Elastasa Pancreática/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Clonación Molecular , Medios de Cultivo Condicionados , Escherichia coli , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/aislamiento & purificación , Humanos , Hidrólisis , Cinética , Leucocitos/enzimología , Macrófagos/citología , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostaglandins G2 and H2 from arachidonic acid. Replacement of the heme group by mangano protoporphyrin IX largely preserves the cyclooxygenase activity, but lowers the steady-state peroxidase activity by 25-fold. Thus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS. A detailed kinetic analysis of the peroxidase reaction using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), EtOOH, and other peroxides as substrates has been carried out to compare the characteristics of PGHS reconstituted with mangano protoporphyrin IX (Mn-PGHS) to those of the native heme enzyme (Fe-PGHS). The rate constant describing the reaction of Mn-PGHS with 15-HPETE to form the oxidized, Mn(IV) intermediate with absorption at 420 nm, exhibits saturable behavior as the 15-HPETE concentration is raised from 10 to 400 microM. This is most likely due to the presence of a second, earlier intermediate between the resting enzyme and the Mn(IV) species. Measurements at high substrate concentrations permitted resolution of the absorbance spectra of the two oxidized Mn-PGHS intermediates. The spectrum of the initial intermediate, assigned to a Mn(V) species, had a line shape similar to that of the later intermediate, assigned to a Mn(IV) species, suggesting that a porphyrin pi-cation radical is not generated in the peroxidase reaction of Mn-PGHS. The rate constant estimated for the formation of the earlier intermediate with 15-HPETE is 1.0 x 10(6) M-1 s-1 (20 degrees C, pH 7.3). A rate constant of 400 +/- 100 s-1 was estimated for the second step in the reaction. Thus, Mn-PGHS reacts considerably more slowly than Fe-PGHS with 15-HPETE to form the first high-valent intermediate, but the two enzymes appear to follow a similar overall reaction mechanism for generation of oxidized intermediates. The difference in rate constants explains the observed lower steady-state peroxidase activity of Mn-PGHS compared with Fe-PGHS.
Asunto(s)
Hemo/metabolismo , Peroxidasa/metabolismo , Fármacos Fotosensibilizantes , Prostaglandina-Endoperóxido Sintasas/metabolismo , Protoporfirinas/metabolismo , Animales , Aspirina/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Hemo/química , Peróxido de Hidrógeno/farmacología , Indometacina/farmacología , Leucotrienos/metabolismo , Peróxidos Lipídicos/metabolismo , Inhibidores de la Lipooxigenasa/metabolismo , Masculino , Prostaglandina H2 , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandinas G/biosíntesis , Prostaglandinas H/biosíntesis , Protoporfirinas/química , Ovinos , Espectrofotometría AtómicaRESUMEN
Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P1' position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of these enzymes show that their respective S1' subsites differ primarily due to the amino acids present at positions 214 and 215. To examine the role that these residues play in determining P1' specificity, the amino acids at these positions in matrilysin have been replaced by those found in stromelysin (MAT: Y214L, MAT:A215V, and MAT:Y214L/A215V). The specificity and activity of MAT:A215V are similar to those of wild type matrilysin. Both MAT:Y214L and MAT:Y214L/A215V, however, have P1' specificities that are more similar to stromelysin than matrilysin. Specifically, these enzymes exhibit an 8- to 9-fold reduction in kcat/KM toward a peptide substrate with Leu in subsite P1' relative to wild type matrilysin. This is predominantly the result of an approximate 5-fold decrease in kcat. The KM values only partially increase toward the value observed for stromelysin. Studies of the pre-steady-state reaction of wild type and mutant matrilysin with substrates with Leu and Tyr residues in the P1' position confirm that the KM values for these reactions reflect KD values for substrate binding. Thus, replacement of a single tyrosine residue in the S1' pocket of matrilysin by leucine alters its P1' specificity to resemble that of stromelysin. In contrast, alteration of the S1' subsite of stromelysin (HFS:L214Y/V215A) to resemble matrilysin increases activity (i.e., higher kcat/KM) toward peptide substrates with both leucine and residues with aromatic side chains in the P1' position with only a partial increase in specificity for Leu. These increases in activity are the result of decreases in the KM values for these reactions.
Asunto(s)
Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Escherichia coli , Humanos , Cinética , Metaloproteinasa 3 de la Matriz , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The zinc contents of samples of human fibroblast collagenase (HFC) purified by different procedures and of samples purified by the same procedure but prepared for analysis by different dialysis protocols have been determined by atomic absorption spectroscopy. Both the purification method and dialysis conditions affect the zinc stoichiometry. Samples purified with and without the use of a zinc-chelate chromatography step and prepared by dialysis against 1 mM CaCl2 had zinc to enzyme ratios of 1.46 and 1.22, respectively. When the first sample was prepared by dialysis against 0 and 10 mM CaCl2, the values changed to 0.15 and 1.94, respectively. Thus, the zinc content of HFC is critically dependent upon the dialysis conditions used to free the enzyme from adventitious metals. This could account for the disparate reports in the literature that give zinc stoichiometries for members of the matrix metalloproteinase (MMP) family of between 1 and 2. The mechanism of inhibition of the one zinc form of HFC by 1,10-phenanthroline (OP) and 4-(2-pyridylazo)resorcinol has been studied in detail. Inhibition by both chelating agents is time dependent and biphasic. There is an initial, instantaneous inhibition characterized by the involvement of a single inhibitor molecule that corresponds to the formation of a ternary complex between the zinc atom, enzyme, and chelator. This is followed by a second, slower phase involving removal of the zinc atom from the enzyme and its chelation by two molecules of inhibitor. Inhibition of four other human MMPs by OP shows similar characteristics and is thought to occur by the same mechanism.
Asunto(s)
Colagenasas/metabolismo , Zinc/metabolismo , Células Cultivadas , Quelantes/farmacología , Colagenasas/química , Humanos , Metaloproteinasa 8 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Fenantrolinas/farmacología , Inhibidores de Proteasas/farmacología , Resorcinoles/farmacología , Espectrofotometría AtómicaRESUMEN
Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Metaloendopeptidasas/aislamiento & purificación , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cromatografía de Afinidad , Cricetinae , Cristalización , Activación Enzimática , Escherichia coli/genética , Humanos , Hidrólisis , Cinética , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Pliegue de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgCl2 is a moderately effective activator, while p-chloromercuribenzoate and NaOCl are poor activators. It is also shown that human matrilysin and human fibroblast-type collagenase can activate pro-HNG by a mechanism that is very similar to that of stromelysin. Initially, these proteinases hydrolyze the Glu40-Met41 bond in the propeptide domain to generate an 88 kDa inactive HNG species. Collagenase also generates a 68 kDa HNG species through hydrolysis of the Ala74-Met75 bond. Ultimately, treatment with either matrilysin, collagenase or trypsin results in the production of a 65 kDa active form of HNG that arises from hydrolysis of the Arg87-Phe88 bond. This is the same active species produced on activation by stromelysin. This cleavage site is downstream of the 'cysteine-switch' residue located at position 80 and releases it, accounting for the permanent activation of the enzyme. These results suggest that matrilysin and collagenase may be physiologically relevant activators of pro-HNG and/or other progelatinase B species. Activation by HgCl2 produces an active 68 kDa enzyme due to autolytic hydrolysis of the Ala74-Met75 bond. This species retains the cysteine switch residue; however, it is shown that it is only active in the continued presence of HgCl2. Removal of the HgCl2 restores latency, indicating that this species is reversibly activated by HgCl2, which functions by complexing the sulfhydryl group of the cysteine switch residue and keeping it dissociated from the active site zinc atom. Thus, in spite of reports to the contrary, the cysteine switch mechanism can account for the latency and activation of pro-HNG.
Asunto(s)
Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Neutrófilos/enzimología , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/química , Gelatinasas/química , Humanos , Metaloproteinasa 3 de la Matriz , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/química , Metaloendopeptidasas/farmacología , Datos de Secuencia Molecular , Peso Molecular , Tripsina/farmacologíaAsunto(s)
Colágeno/química , Colagenasas/metabolismo , Hemopexina/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Colágeno/metabolismo , Fibroblastos/enzimología , Humanos , Cinética , Metaloproteinasa 8 de la Matriz , Datos de Secuencia Molecular , Neutrófilos/enzimología , Oligopéptidos/síntesis química , Oligopéptidos/química , Desnaturalización Proteica , Ratas , Tendones/metabolismo , TermodinámicaRESUMEN
Promatrilysin expressed in Escherichia coli and Chinese hamster ovary cells contains 2.36 +/- 0.19 and 2.13 +/- 0.39 moles of zinc per mole of protein, respectively, while the activated enzyme contains 2.22 +/- 0.21. The catalytic domain of stromelysin-1 expressed in E. coli contains 2.22 +/- 0.11. Thus these matrix metalloproteinases contain two metal binding sites at which zinc is bound firmly and possibly a third site at which it is bound weakly. Promatrilysin and matrilysin do not contain significant amounts of Fe, Cu, Mn, or Ni. All known matrix metalloproteinases have a sequence homologous to the zinc binding site of astacin, HExxHxxGxxH, suggesting that one of the zinc sites is catalytic in agreement with the known inhibition of these enzymes by chelators.
Asunto(s)
Apoenzimas/química , Precursores Enzimáticos/química , Metaloendopeptidasas/química , Zinc/análisis , Secuencia de Aminoácidos , Animales , Apoenzimas/biosíntesis , Sitios de Unión , Células CHO , Clonación Molecular , Cricetinae , Precursores Enzimáticos/biosíntesis , Escherichia coli , Humanos , Metaloproteinasa 3 de la Matriz , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The stopped-flow fluorescence technique has been used to study the hydrolysis of 10 dansyl peptides by thermolysin. The origin of the fluorescence changes observed during the reactions has been investigated in detail. Depending on the substrate and the excitation wavelength, the dansyl fluorescence changes observed arise either from energy transfer (maximal at lambda ex = 230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from both sources. These excitation (maximal at lambda ex = 245 and 340 nm) of the free substrate and product, or from both sources. These two types of fluorescence signals reflect the concentrations of ESi and free substrate, respectively. Both types of fluorescence changes have been used to monitor the reaction progress, and different mathematical formalisms have been used to determine the kinetic parameters for the reactions with results that are in good agreement. The efficiency of Trp quenching by a series of five dansyl tripeptides is shown to be related to the fractional saturation of enzyme and follows the KM-1 values for the substrates. The quenching efficiency for a dansyl tetrapeptide is weaker due to the greater distance between the dansyl group and the Trp-115 donor in thermolysin. On the basis of these studies, substrates capable of supporting more detailed kinetic studies of thermolysin have been identified.
Asunto(s)
Compuestos de Dansilo/química , Péptidos/metabolismo , Termolisina/química , Secuencia de Aminoácidos , Hidrólisis , Cinética , Datos de Secuencia Molecular , Péptidos/química , Espectrometría de Fluorescencia , Especificidad por Sustrato , Termolisina/antagonistas & inhibidoresRESUMEN
The effect of sodium halide salts on the hydrolysis of three of the dansyl (Dns) peptide substrates described in the previous paper (Yang & Van Wart, 1994) by thermolysin have been studied. Increasing concentrations of sodium chloride decrease the KM value for the hydrolysis of the tripeptides Dns-Gly-Phe-Ala and Dns-Ala-Phe-Ala but leave kcat unaltered. This kinetic behavior is described by a nonessential activation mechanism in which chloride binds preferentially to the enzyme-substrate complex. Similar trends are found for the sodium bromide and fluoride salts. In contrast, sodium chloride decreases both KM and kcat almost equally for the hydrolysis of Dns-Ala-Ala-Phe-Ala, leaving kcat/KM unchanged. Thus, chloride is an uncompetitive inhibitor of this substrate. Molecular modeling studies have been carried out in order to explain the effect of chloride on the binding of these dansyl peptides. The decrease in KM for the hydrolysis of all three substrates is attributed to an interaction of chloride with Arg-203 located in the active site to stabilize the enzyme-substrate complexes. The differential effect of chloride on the kcat values for the hydrolysis of the dansyl tripeptides vs dansyl tetrapeptide is related to differences in binding on the Pn side of the substrates. The tripeptides are predicted to bind to the active site of thermolysin in a single low-energy conformation. However, there are two populations of low-energy binding modes for the tetrapeptide, one of which is believed to be a more productive binding mode.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aniones/metabolismo , Compuestos de Dansilo/metabolismo , Péptidos/metabolismo , Termolisina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Compuestos de Dansilo/química , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Compuestos de Sodio/química , Compuestos de Sodio/metabolismo , Especificidad por SustratoRESUMEN
The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagenase), and putative metalloproteinase (PUMP or matrilysin) have been examined by measuring the rate of hydrolysis of over 50 synthetic oligopeptides covering the P4 through P4' subsites of the substrate. The peptides investigated in this paper were those employed in our previous study which systematically examined the sequence specificity of human fibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J. Biol. Chem. 266, 6747]. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured under first-order conditions ([S0] << KM), and kcat/KM values have been calculated from the initial rates. The specificities of these five metalloproteinases are similar, but distinct, with the largest differences occurring at subsites P1, P1', and P3'. The specificities of the two gelatinases are the most similar to each other. They tolerate only small amino acids such as Gly and Ala in subsite P1. In contrast, larger residues such as Met, Pro, Gln, and Glu are also accommodated well by PUMP. All five enzymes prefer hydrophobic, aliphatic residues in subsite P1'. PUMP exhibits a stronger preference for Leu in this subsite than is shown by the other enzymes. The P3' subsite specificities of the gelatinases and collagenases are very similar but different from those of PUMP which particularly prefers Met in this position. The specificity data from this study allow the design of optimized substrates and selective inhibitors for these metalloproteinases.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Matriz Extracelular , Metaloendopeptidasas/metabolismo , Oligopéptidos/metabolismo , Agrecanos , Secuencia de Aminoácidos , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Colágeno/metabolismo , Gelatinasas , Humanos , Cinética , Lectinas Tipo C , Metaloproteinasa 7 de la Matriz , Datos de Secuencia Molecular , Peso Molecular , Proteoglicanos/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The heme coordination of ovine prostaglandin H synthase (PGHS) has been characterized by EPR, magnetic circular dichroism, resonance Raman, and optical spectroscopies. The EPR spectrum of ferric PGHS is consistent with an equilibrium mixture of high-spin and low-spin heme species. Both species disappear on reaction of the synthase with hydroperoxides. The high-spin to low-spin interconversion is temperature- and concentration-dependent. Correlation between the axial and rhombic ligand fields of the low-spin heme species suggests that it has bishistidine axial ligation. Magnetic circular dichroism spectra of PGHS also show a temperature-dependent spin transition. Resonance Raman spectra indicate that the enzyme exists as a mixture of six-coordinate low-spin and six-coordinate high-spin ferric heme species. No Raman bands attributable to five-coordinate high-spin heme species are detectable. The magnetic circular dichroism spectra of the fluoride, azide, cyanide, and imidazole derivatives of PGHS resemble those of the corresponding metmyoglobin derivatives and are very different from those of the catalase derivatives. EPR spectra of the imidazole derivative of these three proteins provide additional evidence that the heme coordination structure of PGHS is similar to that of metmyoglobin rather than that of catalase. The midpoint potential of the PGHS Fe(III)/Fe(II) pair is in the range observed for hemeproteins with mono- or bishistidine coordination. These data provide a convincing case that the axial heme ligands of PGHS-1 are a pair of histidine residues, with the distal histidine weakly associated and possibly exchangeable with a weak-field ligand.