RESUMO
Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.
Assuntos
Escherichia coli/enzimologia , Edição de RNA , RNA de Transferência de Valina/metabolismo , Valina-tRNA Ligase/metabolismo , Regiões 3' não Traduzidas/metabolismo , Acilação , Sítios de Ligação , Escherichia coli/genética , Hidrólise , Isoleucina/metabolismo , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Aminoacilação de RNA de Transferência , Valina-tRNA Ligase/antagonistas & inibidoresRESUMO
SB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl, leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS.
Assuntos
Antibacterianos/síntese química , Inibidores Enzimáticos/síntese química , Indenos/química , Indenos/síntese química , Isoleucina-tRNA Ligase/antagonistas & inibidores , Piridinas , Sulfonamidas/química , Sulfonamidas/síntese química , Compostos de Enxofre , Valina-tRNA Ligase/antagonistas & inibidores , Alcaloides/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indenos/farmacologia , Cinética , Fígado/enzimologia , Testes de Sensibilidade Microbiana , Ratos , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Marcadores de Afinidade/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Metionina tRNA Ligase/metabolismo , Valina-tRNA Ligase/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cinética , Metionina tRNA Ligase/antagonistas & inibidores , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Tripsina , Valina-tRNA Ligase/antagonistas & inibidoresRESUMO
The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Sulfato de Amônio/farmacologia , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Valina/metabolismo , Valina-tRNA Ligase/metabolismo , Sulfato de Amônio/administração & dosagem , Catálise , Fenômenos Químicos , Físico-Química , Relação Dose-Resposta a Droga , Eletroquímica , Cinética , Vírus do Mosaico/genética , RNA Fúngico/metabolismo , RNA Viral/metabolismo , Saccharomyces cerevisiae/enzimologia , Cloreto de Sódio/farmacologia , Valina-tRNA Ligase/antagonistas & inibidoresRESUMO
The interaction of aminoacyl-tRNA synthetase with RNA and polyanions was studied. The inhibition of the enzymes by polyU, polyI and heparin was demonstrated. It was found that this interaction is of limited specificity and is typical of single-stranded RNAs which possess no orderly secondary structure as well as of other polyanions possessing similar polyelectrolytic properties. Data from kinetic analysis and lysyl-tRNA synthetase modification by pyridoxal phosphate are suggestive of participation of the tRNA binding site in the enzyme interaction with polyanions.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Fígado/enzimologia , Polímeros/metabolismo , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Animais , Arginina-tRNA Ligase/antagonistas & inibidores , Arginina-tRNA Ligase/metabolismo , Heparina/farmacologia , Cinética , Lisina-tRNA Ligase/antagonistas & inibidores , Lisina-tRNA Ligase/metabolismo , Poli I/farmacologia , Poli U/farmacologia , Polieletrólitos , Coelhos , Valina-tRNA Ligase/antagonistas & inibidores , Valina-tRNA Ligase/metabolismoRESUMO
The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.
Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Patulina/farmacologia , Piranos/farmacologia , Animais , Arginina-tRNA Ligase/antagonistas & inibidores , Aspartato-tRNA Ligase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Fígado/enzimologia , Camundongos , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Valina-tRNA Ligase/antagonistas & inibidoresRESUMO
The reductive activation of a valyl-tRNA synthetase from yeast is strongly inhibited by 1-30 microM unsaturated fatty acids, and the inhibition is antagonized by 10-100 microM saturated fatty acids. Diethylstilbestrol also inhibits the activation. The possibility that unesterified palmitoleic and oleic acids are bona fide regulatory effectors is supported by a dramatic inverse relation between their cellular content and the growth rate of commercial bakers' yeast. An increase in the ratio of unsaturated to saturated acids with slowing growth in a laboratory strain, S288C, also supports the regulatory hypothesis. The free fatty acids are extracted into slightly acidified 50% alcohol together with traces of numerous novel lipophilic substances. One of these is suggested to function as a cofactor in conjunction with a heat-stable polypeptide that activates valyl-tRNA synthetase.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Ácidos Graxos Insaturados/fisiologia , Saccharomyces cerevisiae/enzimologia , Valina-tRNA Ligase/metabolismo , Ativação Enzimática , Cinética , Oxirredução , Saccharomyces cerevisiae/crescimento & desenvolvimento , Relação Estrutura-Atividade , Valina-tRNA Ligase/antagonistas & inibidoresRESUMO
S-Adenosylhomocysteine inhibits ATP-PPi exchange and tRNA aminoacylation reactions catalysed by pure lupin valyl-tRNA synthetase. The reactions catalysed by phenylalanyl-tRNA, tyrosyl-tRNA, seryl-tRNA, arginyl-tRNA and tryptophanyl-tRNA synthetases are not inhibited by S-adenosylhomocysteine. The inhibition is proportional to first power of S-adenosylhomocysteine concentration. S-Adenosylhomocysteine is a competitive inhibitor of the lupin valyl-tRNA synthetase with respect to ATP and valine in the ATP-PPi exchange reaction, noncompetitive with respect to ATP and valine and uncompetitive with respect to tRNA in the tRNA aminoacylation reaction. Inhibition constant for S-adenosylhomocysteine determined from the reciprocal of Km for tRNA vs. the inhibitor concentration (at saturating ATP and not saturating valine concentration) is 0.35 mM. Inhibition constants for S-adenosylhomocysteine determined from the ATP-PPi exchange are 0.21 mM at saturating valine concentration with variable ATP and 0.33 mM at saturating ATP concentration with variable valine. The data are consistent with the binding of S-adenosylhomocysteine to the valyl adenylate site on the enzyme.
Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Homocisteína/análogos & derivados , S-Adenosil-Homocisteína/farmacologia , Sementes/enzimologia , Valina-tRNA Ligase/antagonistas & inibidores , Cinética , Relação Estrutura-AtividadeRESUMO
Phosphonomethyl analogues of glycyl phosphate and valyl phosphate, i.e. NH2-CHR-CO-CH2-PO(OH)2, were synthesized and esterified with adenosine to give analogues of aminoacyl adenylates. The interaction of these adenylate analogues with valyl-tRNA synthetase from Escherichia coli was studied by fluorescence titration. The analogue of valyl phosphate has an affinity for the enzyme comparable with that of valine, but that of valyl adenylate is bound much less tightly than either valyl adenylate or corresponding derivative of valinol. The affinity of the analogue of glycyl adenylate was too low to be measured. We conclude that this enzyme interacts specifically with both the side chain and the anhydride linkage of the adenylate intermediate.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Organofosfonatos/síntese química , Monofosfato de Adenosina/síntese química , Monofosfato de Adenosina/farmacologia , Sítios de Ligação , Estabilidade de Medicamentos , Glicina/farmacologia , Cinética , Organofosfonatos/farmacologia , Valina/farmacologia , Valina-tRNA Ligase/antagonistas & inibidoresAssuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Isoleucina-tRNA Ligase/antagonistas & inibidores , Oligonucleotídeos/farmacologia , Oligorribonucleotídeos/farmacologia , RNA de Transferência , Valina-tRNA Ligase/antagonistas & inibidores , Sequência de Bases , Escherichia coli/enzimologia , Marcação por Isótopo , Cinética , Relação Estrutura-Atividade , TrítioRESUMO
Nineteen analogs of ATP have been tested in the aminoacylation of valyl-tRNA, isoleucyl tRNA and tyrosyl-tRNA synthetases from baker's yeast. Four compounds are substrates for valyl tRNA and two for isoleucyl-tRNA synthetase, but there is no modified substrate for the tyrosyl tRNA synthetase. There is one inhibitor for valyl-tRNA synthetase, eight compounds inhibit isoleucyl-tRNA synthetase and two compounds inhibit tyrosyl-tRNA synthetase. Their Km and Ki and V values have been determined. The substrate specificity shows that positions 2, 6, 7, 8, 9, 2', and 3' of ATP are important for catalytic action of these aminoacyl-tRNA synthetases.