RESUMO
p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.
Assuntos
Cresóis/farmacologia , Dopamina beta-Hidroxilase/antagonistas & inibidores , Tirosina , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Homologia de Sequência do Ácido Nucleico , TripsinaRESUMO
Succinyl-CoA:tetrahydrodipicolinate-N-succinyltransferase is a key enzyme in the biosynthesis of diaminopimelic acid (DAP), a component of the cell wall peptidoglycan of nearly all bacteria. This enzyme converts the cyclic precursor tetrahydrodipicolinic acid (THDPA) to a succinylated acyclic product. L-2-Aminopimelic acid (L-1), an acyclic analogue of THDPA, was found to be a good substrate for this enzyme and was shown to cause a buildup of THDPA in a cell-free enzyme system but was devoid of antibacterial activity. Incorporation of 1 into a di- or tripeptide yielded derivatives that exhibited antibacterial activity against a range of Gram-negative organisms. Of the five peptide derivatives tested, (L-2-aminopimelyl)-L-alanine (6) was the most potent. These peptides were shown to inhibit DAP production in intact resting cells. High levels (30 mM) of 2-aminopimelic acid were achieved in the cytoplasm of bacteria as a result of efficient uptake of the peptide derivatives through specific peptide transport systems followed, presumably, by cleavage by intracellular peptidases. Finally, the antibacterial activity of these peptides could be reversed by DAP or a DAP-containing peptide. These results demonstrate that the peptides containing L-2-aminopimelic acid exert their antibacterial action by inhibition of diaminopimelic acid biosynthesis.
Assuntos
Diamino Aminoácidos/antagonistas & inibidores , Ácido Diaminopimélico/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Peptídeos/farmacologia , Ácidos Pimélicos/farmacologia , Aciltransferases/antagonistas & inibidores , Bacillus/efeitos dos fármacos , Bacillus/metabolismo , Fenômenos Químicos , Química , Ácido Diaminopimélico/biossíntese , Ácido Diaminopimélico/farmacologia , Enterobacter/efeitos dos fármacos , Enterobacter/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Lisina/farmacologia , Peptídeos/síntese química , Ácidos Pimélicos/síntese química , Ácidos Pimélicos/metabolismoRESUMO
Structure-activity relationships (SAR) were determined for novel multisubstrate inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by examining the effects upon in vitro inhibitory potencies resulting from structural changes at the copper-binding region of inhibitor. Attempts were made to determine replacement groups for the thione sulfur atom of the prototypical inhibitor 1-(4-hydroxybenzyl)imidazole-2-thione described previously. The synthesis and evaluation of oxygen and nitrogen analogues of the soft thione group demonstrated the sulfur atom to be necessary for optimal activity. An additional series of imidazole-2-thione relatives was prepared in an effort to probe the relationship between the pKa of the ligand group and inhibitory potency. In vitro inhibitory potency was shown not to correlate with ligand pKa over a range of approximately 10 pKa units, and a rationale for this is advanced. Additional ligand modifications were prepared in order to explore bulk tolerance at the enzyme oxygen binding site and to determine the effects of substituting a six-membered ligand group for the five-membered imidazole-2-thione ligand.
Assuntos
Dopamina beta-Hidroxilase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Animais , Sítios de Ligação , Bovinos , Fenômenos Químicos , Química , Físico-Química , Cobre/metabolismo , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Ligantes , Piridinas/síntese química , Piridinas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/farmacologia , Tionas/síntese química , Tionas/farmacologia , Triazóis/farmacologiaRESUMO
1-Aralkylimidazole-2-thiones have been shown to be potent multisubstrate inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1). In the present study, a series of 1-benzylimidazole-2-thiones was prepared to explore the effects of substitution in the benzyl ring on the inhibition of DBH. A detailed structure-activity relationship for in vitro activity was discovered and this was shown by a modified Hansch analysis to correlate (r = 0.91) with four key structural features of the benzyl ring: the presence of a hydroxyl at the 4-position, molar refractivity at the 3-, 4-, and 5-positions, inductive effects of the substituents at the 3-, 4-, and 5-positions, and pi-electron density. The affinity (Kis) of eight substituted inhibitors for DBH was shown to correlate (r = 0.75) with the affinity (KD) of comparably substituted tyramines for the ternary DBH-oxygen-tyramine complex. This correlate is used to support the hypothesis that binding of inhibitor to DBH occurs in a fashion that mimics the binding of tyramine substrates. The most potent inhibitors were selected for study in vivo in the spontaneously hypertensive rat model of hypertension. The changes in vascular dopamine and norepinephrine levels that resulted from oral administration of the inhibitors corresponded to the observed reduction in mean arterial blood pressure. A divergence between in vitro potency and in vivo efficacy upon oral dosing was noted and is suggested to result from an in vivo metabolic conjugation of the phenolic group of inhibitor.
Assuntos
Dopamina beta-Hidroxilase/antagonistas & inibidores , Imidazóis/síntese química , Fenetilaminas/metabolismo , Animais , Sítios de Ligação , Pressão Sanguínea/efeitos dos fármacos , Imidazóis/farmacologia , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Masculino , Ligação Proteica , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Tionas/síntese química , Tionas/farmacologiaRESUMO
The synthesis and characterization of some 1-(phenylalkyl)imidazole-2-thiones as a novel class of "multisubstrate" inhibitors of dopamine beta-hydroxylase (DBH) are described. These inhibitors incorporate structural features that resemble both tyramine and oxygen substrates, and as evidenced by steady-state kinetics, they appear to bind both the phenethylamine binding site and the active site copper atom(s) in DBH. A series of structural congeners that incorporate different bridging chain lengths between the phenyl ring (dopamine mimic) and the imidazole-2-thione group (oxygen mimic) define the optimum distance for inhibitory potency and the likely intersite distance in the DBH active site. Additional bridging analogues were prepared to determine the active site bulk tolerance and the effects of heteroatom replacement.
Assuntos
Dopamina beta-Hidroxilase/antagonistas & inibidores , Imidazóis/síntese química , Animais , Bovinos , Imidazóis/farmacologia , Indicadores e Reagentes , Cinética , Espectroscopia de Ressonância Magnética , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Tionas/síntese química , Tionas/farmacologiaRESUMO
5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/síntese química , Isatina/análogos & derivados , Isatina/síntese química , Sulfonamidas/síntese química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 7 , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Humanos , Isatina/química , Isatina/farmacologia , Células Jurkat , Cinética , Camundongos , Modelos Moleculares , Conformação Molecular , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Antiasmáticos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Animais , Antiasmáticos/síntese química , Antiasmáticos/metabolismo , Antiasmáticos/toxicidade , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/toxicidade , Ligação Competitiva , Temperatura Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Broncoconstrição/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ácidos Cicloexanocarboxílicos/síntese química , Ácidos Cicloexanocarboxílicos/metabolismo , Ácidos Cicloexanocarboxílicos/toxicidade , Cães , Ácido Gástrico/metabolismo , Cobaias , Humanos , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Nitrilas , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/toxicidade , Pirrolidinonas/síntese química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Pirrolidinonas/toxicidade , Coelhos , Proteínas Recombinantes/antagonistas & inibidores , Rolipram , Estereoisomerismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Vômito/induzido quimicamenteRESUMO
In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.
Assuntos
Apoptose , Caspases/fisiologia , Túbulos Renais Proximais/citologia , Transativadores , Clorometilcetonas de Aminoácidos/farmacologia , Proteínas do Citoesqueleto/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Ácido Okadáico/farmacologia , Oligopeptídeos/farmacologia , beta Catenina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.
Assuntos
Apoptose , Caspases/fisiologia , Condrócitos/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 3 , Inibidores de Caspase , Linhagem Celular , Colágeno/genética , Humanos , Oligopeptídeos/farmacologia , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
The synthesis of a new 8-spin-labeled analog of AMP, 8-[[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)carbamoyl]methyl]thio]adenosine 5'-phosphate (8-slAMP), is described. The procedure is facile and results in high yields. 8-slAMP is a competitive inhibitor of AMP nucleosidase with a Ki of 19 microM as compared to a Km of 100 microM for AMP. The analog is not a substrate for the enzyme and does not displace MgATP2- from the allosteric sites under the usual assay conditions. The EPR spectrum of the bound spin probe reveals a highly immobilized nitroxide group. Binding studies with 8-slAMP at 8 degrees C indicate three independent binding sites (Kd = 1.4 microM) per molecule of enzyme (Mr = 320,000). These properties make 8-slAMP a good spin probe for AMP nucleosidase. The analog may also be useful for other proteins known or suspected of binding AMP analogs in a syn conformation.
Assuntos
Monofosfato de Adenosina/análogos & derivados , N-Glicosil Hidrolases , Marcadores de Spin , Monofosfato de Adenosina/farmacologia , Azotobacter/enzimologia , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , N-Glicosil Hidrolases/metabolismo , Ligação ProteicaRESUMO
The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.
Assuntos
Medula Suprarrenal/enzimologia , Cresóis/farmacologia , Dopamina beta-Hidroxilase/antagonistas & inibidores , Fenóis/farmacologia , Animais , Bovinos , Grânulos Cromafim/enzimologia , Hidroxilação , Cinética , Relação Estrutura-Atividade , Trítio , Tiramina/farmacologiaRESUMO
The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Azotobacter/enzimologia , N-Glicosil Hidrolases/metabolismo , Adenina/farmacologia , Monofosfato de Adenosina/análise , Radioisótopos de Carbono , Cinética , Ribosemonofosfatos/farmacologia , TermodinâmicaRESUMO
Considerable interest has been generated in the potential utility of phosphodiesterase (PDE) IV inhibitors as a novel class of anti-asthmatic agents. Because a detailed understanding of the molecular and biochemical characteristics of any molecular target of interest provides a key ingredient for rational drug design, we cloned a cDNA encoding a PDE IV (hPDE IV) from a human monocyte library and expressed, purified and characterized the recombinant gene product. Purified hPDE IV has kinetic characteristics consistent with native PDE IV isolated from tissue sources. In addition, it is inhibited by rolipram (Ki = 60 nM) and other archetypical PDE IV-selective inhibitors. Purified hPDE IV also contains a high affinity binding site for rolipram (Kd = 2 nM), although the precise relationship between this site and the cAMP catalytic site is not clear. In other studies in which the regulation of PDE IV expression was examined in U937 cells, a human monocytic cell line, prolonged treatment with salbutamol was shown to induce an increase in the activity of PDE IV. This up-regulation of PDE IV activity appears to be mediated by cAMP and occurs at the transcriptional or pretranscriptional level. As a consequence of PDE IV up-regulation, the sensitivity of U937 cells to the inhibitory effects of adenylyl cyclase activators on cell function is greatly diminished. If such regulation of PDE IV occurs in inflammatory cells in vivo, it could have implications for the therapeutic use of beta-adrenoceptor agonists. Specifically, induction of PDE IV activity in asthmatics being treated with beta-adrenoceptor agonists could result in a heterologous desensitization of inflammatory cells to endogenous anti-inflammatory agents (e.g., epinephrine, prostaglandin E2).
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Diester Fosfórico Hidrolases/química , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Cinética , Inibidores de Fosfodiesterase/metabolismo , Diester Fosfórico Hidrolases/isolamento & purificação , Diester Fosfórico Hidrolases/fisiologia , Pirrolidinonas/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rolipram , Regulação para Cima/fisiologiaRESUMO
beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dopamina beta-Hidroxilase/antagonistas & inibidores , Tiramina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Indicadores e Reagentes , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Ligação Proteica , Estereoisomerismo , Tiramina/síntese química , Tiramina/farmacocinéticaRESUMO
The synthesis and kinetics characterization of a new class of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) inhibitor, 1-(4-hydroxybenzyl)imidazole-2-thiol, is reported. These inhibitors, which incorporate a phenethylamine substrate mimic and an oxygen mimic into a single molecule, exhibit both the kinetic properties and the potency (Kis approximately 10(-9) M) expected for a multisubstrate inhibitor and are therefore classified as such. Steady-state kinetic experiments with these multisubstrate inhibitors and their substructural analogues support the recently proposed pH-dependent changes in substrate binding order [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3106] and a mechanism whereby the inhibitor binds specifically to the reduced Cu+ form of enzyme at both the phenethylamine substrate site and the active-site copper atom(s). A Yonetani-Theorell double-inhibition experiments indicates mutually exclusive binding of the inhibitor substructures p-cresol and 1-methylimidazole-2-thiol to suggest an extremely short intersite distance between the phenethylamine binding site and the active-site copper atom(s).
Assuntos
Dopamina beta-Hidroxilase/antagonistas & inibidores , Imidazóis/síntese química , Medula Renal/enzimologia , Animais , Bovinos , Grânulos Cromafim/enzimologia , Imidazóis/farmacologia , Indicadores e Reagentes , Cinética , Matemática , Relação Estrutura-AtividadeRESUMO
A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.
Assuntos
Dopamina beta-Hidroxilase/genética , Glândulas Suprarrenais/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA/genética , Glicosilação , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Homologia de Sequência do Ácido NucleicoRESUMO
X-ray absorption edge and extended X-ray absorption fine structure (EXAFS) spectra are reported for the Cu(I) and Cu(II) forms of bovine dopamine beta-hydroxylase (DBH; EC 1.14.17.1) and for the Cu(I) form of DBH bound either to tyramine substrate or to a multisubstrate inhibitor [Kruse, L. I., DeWolf, W. E., Jr., Chambers, P. A., & Goodhart, P. J. (1986) Biochemistry 25, 7271-7278]. A significant change in the structure of the copper sites occurs upon ascorbate-mediated reduction of Cu(II) DBH to the Cu(I) form. While the average Cu(II) site most likely consists of a square-planar array of four (N,O)-containing ligands at 1.98 A, the average Cu(I) site shows a reduction in (N,O) coordination number (from approximately 4 to approximately 2) and the addition of a S-containing ligand at 2.30 A. No change in the average Cu(I) ligand environment accompanies binding of tyramine substrate, whereas binding of a multisubstrate inhibitor, 1-(3,5-difluoro-4-hydroxybenzyl)-1H-imidazole-2(3H)-thione, causes an increase in the Cu-S coordination, consistent with inhibitor binding to the Cu(I) site through the S atom. Although excellent signal-to-noise ratio in the EXAFS spectra of ascorbate-reduced DBH facilitated analysis of outer-shell scattering for a Cu..Cu interaction, the presence of a binuclear site could not be proven or disproven due to interference from Cu...C scattering involving the carbons of imidazole ligands.
Assuntos
Cobre , Dopamina beta-Hidroxilase , Medula Suprarrenal/enzimologia , Animais , Bovinos , Dopamina beta-Hidroxilase/antagonistas & inibidores , Dopamina beta-Hidroxilase/isolamento & purificação , Oxirredução , Análise Espectral , Raios XRESUMO
The novel dopamine beta-hydroxylase (D beta H) inhibitor SK&F 102698 was characterized in vitro with soluble enzyme from bovine adrenal medulla and in vivo by measuring the dopamine/norepinephrine (DA/NE) ratio in the mesenteric artery, heart and brains of spontaneously hypertensive rats (SHR). SK&F 102698 was a potent D beta H inhibitor with an IC50 of 1.2 microM on crude enzyme and had a Ki value of 40 nM on purified enzyme. SK&F 102698 produced a dose-dependent fall in NE and a dose-dependent increase in DA in the vasculature of SHR after p.o. administration. Elevation of the vascular DA/NE ratio was observed within 0.5 hr after administration. Peak effects were observed at 12 hr and values were still significantly increased at 18 hr. The rise in the DA/NE ratio of the blood vessels correlated with the fall in blood pressure following the first 4 hr after SK&F 102698. SK&F 102698 inhibited SHR heart D beta H and elevated the myocardial DA/NE ratio approximately 2.4-fold. SK&F 102698 also caused a dose-dependent increase in the whole brain DA/NE ratio of SHR. Catecholamine levels were also studied in six discrete brain regions and SK&F 102698 produced the greatest increase in the DA/NE ratio in the cerebellum, brain stem and midbrain regions, whereas the striatum was the region least affected. No overt sedation was observed in the rats. Further study with SK&F 102698 is warranted to better explore the role of DA and D beta H in pathophysiology, and to determine whether this drug or a congener D beta H inhibitor will be a useful therapeutic agent in humans.