RESUMO
Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.
Assuntos
Amino Açúcares/biossíntese , Streptomyces/enzimologia , Transaminases/química , Amino Açúcares/química , Clonagem Molecular , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Estrutura Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fosfato de Piridoxal/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Transaminases/genética , Transaminases/metabolismoRESUMO
ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 microM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K(d)) of 44, 110, and 637 microM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP >or= ATPgammaS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). alphabeta-Methylene-ATP (alphabeta-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK(a)) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)(Ca)/P(Na) = 5.32], but not to chloride (P(Cl)/P(Na) = 0.03) or N-methyl-D-glucamine (NMDG) (P(NMDG)/P(Na) = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between -90 and -50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (tau) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X(2).
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Técnicas de Patch-Clamp , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Receptores Purinérgicos P2X , Suramina/metabolismoRESUMO
Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.
Assuntos
Guanosina Trifosfato/metabolismo , Fosfato de Piridoxal/metabolismo , Coloração e Rotulagem/métodos , Transducina/química , Transducina/metabolismo , Animais , Sítios de Ligação , Boratos/farmacologia , Bovinos , Inibidores Enzimáticos/farmacologia , Olho/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanilil Imidodifosfato/antagonistas & inibidores , Guanilil Imidodifosfato/metabolismo , Ligantes , Luz , Fosfatos/farmacologia , Compostos de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Piridoxal/farmacologia , Fosfato de Piridoxal/farmacologia , TrítioRESUMO
Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in choanomastigotes of Crithidia fasciculata by DEAE-cellulose chromatography. The three isoforms (named SHMT I, II, and III) presented small differences in charge and molecular weight. Digitonin treatment of intact cells suggested that SHMT III is cytosolic, whereas the other two isoforms are particle bound, one being mitochondrial (SHMT I) and the other one very likely glycosomal (SHMT II). The three SHMT isoforms were purified to homogeneity, and their physicochemical and kinetic properties studied. Determination of their native and subunit molecular masses revealed that all of them have a tetrameric structure. The three isoforms were shown to be PLP-dependent enzymes after L-cysteine and hydroxylamine hydrochloride treatments. They showed similar pH optima, bimodal kinetics for L-serine and Michaelis-Menten kinetics for THF.
Assuntos
Crithidia fasciculata/enzimologia , Glicina Hidroximetiltransferase/isolamento & purificação , Animais , Compartimento Celular , Citosol/enzimologia , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/metabolismo , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Mitocôndrias/enzimologia , Peso Molecular , Organelas/enzimologia , Fosfato de Piridoxal/metabolismo , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.
Assuntos
Arginina , Cisteína , Escherichia coli/enzimologia , Lisina , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Sítios de Ligação , Diacetil/farmacologia , Glioxal/análogos & derivados , Glioxal/farmacologia , Cinética , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Pirenos/farmacologia , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.
Assuntos
Carboxiliases/análise , Fígado/enzimologia , Nippostrongylus/enzimologia , Fosfato de Piridoxal/metabolismo , Transaminases/análise , Animais , RatosRESUMO
1. The action of hexachlorobenzene (HCB) on hepatic ferrochelatase was investigated. 2. A direct action of HCB, pentachlorophenol, porphyrins and haem on this enzyme activity was discarded. 3. In HCB porphyric liver there is probably an activator tightly bound to the enzyme. 4. Pyridoxal phosphate (PPL) may be a cofactor of ferrochelatase from both normal and porphyric rats. 5. The PPL would be involved in the binding site of Fe2+ or at least in the approaching of Fe2+ to the active site of the enzyme. 6. The differences found between normal and porphyric preparations could be attributed to conformational changes elicited by the HCB.
Assuntos
Ferroquelatase/metabolismo , Hexaclorobenzeno/farmacologia , Fígado/enzimologia , Porfirias/enzimologia , Animais , Sítios de Ligação , Cromatografia em Gel , Cobre/farmacologia , Sulfato de Cobre , Feminino , Heme/farmacologia , Temperatura Alta , Pentaclorofenol/farmacologia , Porfirias/induzido quimicamente , Porfirinas/farmacologia , Conformação Proteica/efeitos dos fármacos , Fosfato de Piridoxal/metabolismo , Ratos , Ratos Endogâmicos , EspectrofotometriaRESUMO
Studies on the decarboxylation of ornithine in Leishmania mexicana have shown that this activity corresponds to a true ornithine decarboxylase rather than to an oxidative decarboxylation or aminotransferase reaction, both of which also give rise to the release of CO2. The stoichiometric relationship between substrate and products has indicated that extracts of L. mexicana were able to catalyse the formation of an unknown compound besides putrescine and CO2. The addition of cycloheximide to cultures of L. mexicana allowed us to demonstrate that ornithine decarboxylase degradation in vivo was extremely slow in this parasite. This remarkable stability of the enzyme is only comparable to that found in Trypanosoma brucei and contrasts with the high turnover rate of ornithine decarboxylases of different mammalian cells.