RESUMO
The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Metaloendopeptidases/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Combinação de Medicamentos , Sinergismo Farmacológico , Ligases/antagonistas & inibidores , Ligases/genética , Ligases/metabolismo , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pirimidinonas/farmacologia , Percepção de Quorum/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/farmacologiaAssuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite/tratamento farmacológico , Terapia Biológica/métodos , Ligases/antagonistas & inibidores , Doenças Pulmonares Intersticiais/tratamento farmacológico , Miosite/tratamento farmacológico , Adulto , Artrite/complicações , Artrite/diagnóstico , Feminino , Humanos , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/diagnóstico , Imageamento por Ressonância Magnética , Miosite/complicações , Miosite/diagnóstico , Radiografia , SíndromeRESUMO
Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.
Assuntos
Antibacterianos/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Furanos/química , Ligases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Ácidos Carboxílicos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Ligases/química , Ligases/metabolismo , Simulação de Dinâmica Molecular , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. METHODS: MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H(37)Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. RESULTS: Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H(37)Rv. CONCLUSIONS: Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent anti-tubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ligases/antagonistas & inibidores , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/antagonistas & inibidores , Extratos Vegetais/farmacologia , Antituberculosos/isolamento & purificação , Colômbia , Colorimetria/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Plantas/químicaRESUMO
Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)-dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z'>or=0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2-namely, dequalinium chloride-inhibits MshC with an IC50 value of 24+/-1 microM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a KD of 0.22 microM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 microg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors.
Assuntos
Cisteína/metabolismo , Dequalínio/análise , Dequalínio/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Glicopeptídeos/metabolismo , Inositol/metabolismo , Ligases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Cinética , Medições Luminescentes , Proteínas Ligantes de Maltose , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1(C61G), does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Proteínas de Transporte/metabolismo , Ligases/metabolismo , Mutação , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Proteína BRCA1/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , DNA Complementar , Dimerização , Humanos , Ligases/antagonistas & inibidores , Dedos de ZincoRESUMO
An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/metabolismo , Dimetil Sulfóxido/farmacologia , Európio/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunoensaio , Cinética , Ligases/antagonistas & inibidores , Metais Terras Raras/metabolismo , Ficocianina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Ubiquitinas/antagonistas & inibidoresRESUMO
The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co-factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition. In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6-hydroxymellein synthase toward cerulenin.
Assuntos
Aciltransferases/metabolismo , Cerulenina/farmacologia , Daucus carota/enzimologia , Ligases/metabolismo , Complexos Multienzimáticos/metabolismo , NADP/metabolismo , Oxirredutases/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/biossíntese , Indução Enzimática , Cinética , Ligases/antagonistas & inibidores , Ligases/biossíntese , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/biossíntese , NAD/farmacologia , NADP/farmacologia , Oxirredução , Oxirredutases/antagonistas & inibidores , Oxirredutases/biossíntese , Extratos Vegetais , Raízes de PlantasRESUMO
We have studied the in vitro formation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) using a partially purified ppGpp synthetase I (PSI) from Escherichia coli BGA8, a polyamine auxotrophic strain. A comparison of the enzyme obtained from polyamine-supplemented or deprived bacteria showed similar requirements for the reaction, Mg+2 optimum levels and sparing effect of spermidine. No differences in the inhibitory effects of tetracycline, puromycin and fusidic acid were detected either. However, a modified subcellular distribution, as well as a larger specific activity and a larger stimulation by streptomycin was observed when PSI was prepared from polyamine-depleted bacteria. The role of ribosome assembly and subunit distribution on the altered properties of the enzyme are discussed.
Assuntos
Escherichia coli/metabolismo , Ligases/metabolismo , Poliaminas/farmacologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ácido Fusídico/farmacologia , Guanosina Tetrafosfato/biossíntese , Ligases/antagonistas & inibidores , Ligases/isolamento & purificação , Magnésio/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Ribossomos/metabolismo , Espermidina/farmacologia , Estreptomicina/farmacologia , Frações Subcelulares/enzimologia , Tetraciclina/farmacologiaRESUMO
The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de novo purine biosynthesis by these three antagonists results in a "complementary stimulation" of de novo pyrimidine biosynthesis.
Assuntos
Azasserina/farmacologia , Compostos Azo/farmacologia , Carbono-Nitrogênio Ligases , Diazo-Oxo-Norleucina/farmacologia , Glutamina/antagonistas & inibidores , Isoxazóis/farmacologia , Leucemia L1210/enzimologia , Nucleotídeos/biossíntese , Oxazóis/farmacologia , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Ligases/antagonistas & inibidores , Camundongos , Espectrofotometria UltravioletaRESUMO
The purD gene of Escherichia coli encoding the enzyme glycinamide ribonucleotide (GAR) synthetase, which catalyzes the conversion of phosphoribosylamine (PRA), glycine, and MgATP to glycinamide ribonucleotide, MgADP, and Pi, has been cloned and sequenced. The protein, as deduced by the structural gene sequence, contains 430 amino acids and has a calculated Mr of 45,945. Construction of an overproducing strain behind a lambda pL promoter allowed a 4-fold purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of the sequence with those of other GAR synthetases confirm the amino acid sequence deduced from the gene sequence. Initial velocity studies and product and dead-end inhibition studies are most consistent with a sequential ordered mechanism of substrate binding and product release in which PRA binds first followed by MgATP and then glycine; Pi leaves first, followed by loss of MgADP and finally GAR. Incubation of [18O]glycine, ATP, and PRA results in quantitative transfer of the 18O to Pi. GAR synthetase is very specific for its substrate glycine.
Assuntos
Carbono-Nitrogênio Ligases , Escherichia coli/enzimologia , Ligases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Glicina/metabolismo , Cinética , Ligases/antagonistas & inibidores , Ligases/isolamento & purificação , Ligases/metabolismo , Dados de Sequência Molecular , Óperon , Isótopos de Oxigênio , Fósforo/metabolismo , Ribosemonofosfatos/metabolismo , Especificidade por SubstratoRESUMO
Evidence was provided that injection of acivicin (25 mg/kg, i.p.) into the rat inactivated brain CTP and GMP synthetases. Under the same circumstances, CTP and GTP concentrations in the rat brain decreased following the decline in the activities of CTP and GMP synthetases. The decrease in enzymic activities and nucleotide concentrations progressed with time. The decline in CTP and GMP synthetase activities and CTP and GTP concentrations caused by acivicin occurred more slowly and to a lesser extent than in liver and hepatoma 3924A. The delay in the expression of acivicin action in the rat brain was attributed to a possible slower entrance of acivicin and the lower concentration than might have been attained in the rat brain. These considerations are based on the rapid disappearance of acivicin from rat plasma noted earlier. The decline in CTP concentration in rat brain might interfere with neuronal function. The decline in GTP concentration might be expressed through the depletion of biopterins which are generated from GTP in the brain. The possible relevance to the biochemical basis of paranoid schizophrenia which occurs reversibly after high-dose acivicin or tiazofurin treatment was discussed.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Encéfalo/metabolismo , Carbono-Nitrogênio Ligases , Isoxazóis/farmacologia , Ligases/antagonistas & inibidores , Oxazóis/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular , Citidina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Ratos , Ratos Endogâmicos ACIRESUMO
The lignans nordihydroguaiaretic acid (NDGA), heminordihydroguaiaretic acid (HNDGA) and norisoguaiacin were found to inhibit formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) and carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) activity from a wide variety of sources. In all cases, NDGA was the most effective inhibitor. Synthetase activity was reduced by half at NDGA concentrations between 0.11 and 0.24 mM. Esterase activity consisted of NDGA-sensitive and NDGA-resistant forms. The sensitive class was half-inhibited by 2-4 microM NDGA. Irreversible inhibition of formyltetrahydrofolate synthetase by NDGA was observed both at low protein concentration (less than 0.2 mg/ml) and at high protein concentration where precipitation of protein was observed. Inhibition of formyltetrahydrofolate synthetase by NDGA arises from a decrease in Vmax and increase in Km for all substrates. In contrast, NDGA affects only the Vmax parameter of the esterase activity. It is suggested that the broad range of enzymes inhibited by NDGA may be a consequence of the amphipathic character of the molecule and the flexibility to accommodate to a variety of binding sites. It is also suggested that the previously reported ability of NDGA to inhibit phagocytosis may be due to the compound's ability to inhibit carboxylesterases.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Ligases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Catecóis/farmacologia , Bovinos , Células Cultivadas , Citoplasma/enzimologia , Cobaias , Humanos , Cinética , Lignanas , Substâncias Macromoleculares , Masoprocol , Camundongos , Microssomos/enzimologia , Naftóis/farmacologia , Extratos Vegetais/farmacologia , Ligação Proteica , RatosRESUMO
A systematic search has been made for inhibitors of L-asparagine synthetase (L-glutamine hydrolyzing, EC 6.3.5.4) from leukemia 5178Y/AR, a rodent neoplasm resistant to the oncolytic enzyme L-asparaginase (EC 3.5.1.1), The classes of chemicals examined in this search included substrate and product analogs, agents capable of reacting with sulfhydryl functions, and a variety of modifiers whose mechanism of interaction with proteins is known. In general, antagonists of L-glutamine and thiol reagents proved to be the most effective inhibitors of L-asparagine synthetase from this tumor source. Within these groups, certain structural prerequisites to inhibition are reported. Attempts to correlate oncolytic potency with enzyme-inhibitory potency were unsuccesful.
Assuntos
Aspartato-Amônia Ligase/antagonistas & inibidores , Leucemia Experimental/enzimologia , Ligases/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Amônia/farmacologia , Animais , Asparaginase/farmacologia , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Difosfatos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos , Glutamina/análogos & derivados , Glutamina/farmacologia , Heparina/farmacologia , Técnicas In Vitro , Isoxazóis/farmacologia , Malonatos/farmacologia , Metais/farmacologia , Compostos de Nitrosoureia/farmacologia , Reagentes de Sulfidrila/farmacologiaAssuntos
Ligases , Mitocôndrias Hepáticas/enzimologia , Sulfatos , Sulfato de Amônio , Animais , Bicarbonatos , Sítios de Ligação , Precipitação Química , Cloratos , Cromatografia em Gel , Cromatografia por Troca Iônica , Coenzima A , Temperatura Baixa , Concentração de Íons de Hidrogênio , Cinética , Ligases/antagonistas & inibidores , Ligases/isolamento & purificação , Molibdênio , Nitratos , Ovalbumina , Piruvatos , Selênio , Espectrofotometria , Ácidos Sulfônicos , Raios UltravioletaAssuntos
Ligases , Saccharomyces/enzimologia , Trifosfato de Adenosina , Sítios de Ligação , Fenômenos Químicos , Química , Computadores , Difosfatos , Retroalimentação , Concentração de Íons de Hidrogênio , Cinética , Ligases/antagonistas & inibidores , Ligases/isolamento & purificação , Magnésio , Metionina , Modelos Biológicos , Nucleosídeos , Fosfatos , Monoéster Fosfórico Hidrolases , Saccharomyces/análise , SelênioRESUMO
1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.