RESUMO
The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)(6)-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a Delta trpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (His)(6)-PurF/Delta trpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (k(cat)/K(M)=0.3 s(-1) M(-1)) was approximately 25-fold more efficient than its ancestor but >10(7)-fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.
Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Amidofosforribosiltransferase/química , Amidofosforribosiltransferase/metabolismo , Evolução Molecular , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Amidofosforribosiltransferase/genética , Amidofosforribosiltransferase/isolamento & purificação , Catálise , Cromatografia Líquida de Alta Pressão , Cinética , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Seleção Genética , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 degrees C. GPAT activity is known to depend upon channeling of NH(3) from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH(3) for synthesis of PRA was found to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90 degrees C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50 degrees C, and remained relatively constant up to 90 degrees C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.
Assuntos
Amidofosforribosiltransferase/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Nucleotídeos de Adenina , Amidofosforribosiltransferase/antagonistas & inibidores , Amidofosforribosiltransferase/genética , Amidofosforribosiltransferase/isolamento & purificação , Amônia , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/isolamento & purificação , Inibidores Enzimáticos , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Nucleotídeos de Guanina , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , TemperaturaRESUMO
The ability of bovine retina to synthesize purines de novo is shown for the first time. Amidophosphoribosyl transferase (EC 2.4.2.14), the enzyme controlling the rate of the process, and phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1), the enzyme regulating the intracellular contents of phosphoribosyl pyrophosphate (PRPP), were purified and characterized. The molecular masses of the enzyme subunits are similar to those of the purified enzyme from the liver. The molecular masses of amidophosphoribosyl transferase, PRPP synthetase catalytic subunit, and two PRPP synthetase-associated proteins are 50, 34, 39, and 41 kD, respectively. The apparent Km values of the enzymes and coenzymes are similar to those of the purified enzymes from the liver. For amidophosphoribosyl transferase, the apparent Km for Gln and PRPP are 0.75 +/- 0.05 and 0.66 +/- 0.09 mM, respectively (the corresponding Vmax values are 59 +/- 3 and 136 +/- 12 nmoles PPi/min per mg protein). For PRPP synthetase, the apparent Km for ribose-5-phosphate and ATP are 37.9 +/- 0.5 and 53 +/- 7 microM, respectively (the corresponding Vmax values are 61 +/- 4 and 52 +/- 3 nmoles PRPP/min per mg protein). The sensitivity of the retinal PRPP synthetase to inhibition by ADP and AMP was significantly lower than that of the enzyme from the liver.
Assuntos
Amidofosforribosiltransferase/química , Amidofosforribosiltransferase/isolamento & purificação , Purinas/biossíntese , Retina/metabolismo , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/isolamento & purificação , Animais , Bovinos , Cromatografia em Agarose , Cromatografia DEAE-Celulose , CinéticaRESUMO
A cDNA encoding human glutamine phosphoribosylpyrophosphate amidotransferase for step one in de novo purine nucleotide synthesis was cloned, sequenced, and expressed in Chinese hamster ovary cells to yield functional enzyme. Enzyme function was dependent upon removal of an 11-amino-acid propeptide. A mutant enzyme having three propeptide amino acid replacements was not processed and was not active. The human genes GPAT, encoding the amidotransferase, and AIRC, encoding a bifunctional enzyme for steps six and seven in the pathway, were cloned and characterized. GPAT and AIRC are closely linked and divergently transcribed from an intergenic region of approximately 625 base pairs. Expression of a luciferase reporter from the GPAT promoter was approximately 3-4-fold higher than from the AIRC promoter. The GPAT gene was mapped to the q12 region of chromosome 4.
Assuntos
Amidofosforribosiltransferase/genética , Carboxiliases/genética , Cromossomos Humanos Par 4 , Nucleotídeos de Purina/biossíntese , Transcrição Gênica , Amidofosforribosiltransferase/isolamento & purificação , Amidofosforribosiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Bacillus subtilis/enzimologia , Sequência de Bases , Southern Blotting , Células CHO , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Galinhas , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA/análise , Escherichia coli/enzimologia , Genes , Ligação Genética , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudogenes , Ratos , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , TransfecçãoAssuntos
Amidofosforribosiltransferase/antagonistas & inibidores , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacologia , Leucemia L1210/enzimologia , Amidofosforribosiltransferase/química , Amidofosforribosiltransferase/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração , Substâncias Macromoleculares , Camundongos , Células Tumorais CultivadasRESUMO
Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides. Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase. FSBA inactivated the amidotransferase with saturation kinetics. Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of [3H] sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of [3H]SBA. Six chymotryptic peptides modified with [3H]SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity. Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by [3H]SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465. Tyr258 was also modified by N3-AMP. Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme. However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP. Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively. Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation. These results localize residues important for catalysis in close proximity to a site for nucleotide binding. Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP. A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition. Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP. A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.
Assuntos
Amidofosforribosiltransferase/metabolismo , Escherichia coli/enzimologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Marcadores de Afinidade/farmacologia , Amidofosforribosiltransferase/antagonistas & inibidores , Amidofosforribosiltransferase/isolamento & purificação , Sequência de Aminoácidos , Azidas/farmacologia , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Retroalimentação , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fosforribosil Pirofosfato/farmacologia , Plasmídeos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleotídeos/farmacologiaRESUMO
The cDNA of human amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned from human hepatoma (HepG2) cDNA library. The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight (Mr) of 57,398, which is consistent with the molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the ATase subunit purified from human placenta. The derived amino acid sequence exhibits 93, 82, 41, 37, and 33% identity with the sequences of rat, chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively. Southern blot analysis suggested that the ATase gene exists as multiple copies. ATase mRNA (3.5 kb) is ubiquitously expressed in various human tissues. Comparison with rat and chicken ATases showed that two cysteine residues for an iron-sulfur cluster were conserved. Four consensus phosphorylation sites for cAMP-dependent protein kinase were found.
Assuntos
Amidofosforribosiltransferase/genética , Amidofosforribosiltransferase/isolamento & purificação , Amidofosforribosiltransferase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Carcinoma Hepatocelular , Cromatografia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Clonagem Molecular , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Durapatita , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Hidroxiapatitas , Neoplasias Hepáticas , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Fases de Leitura Aberta , Placenta/enzimologia , Reação em Cadeia da Polimerase , Gravidez , Células Tumorais CultivadasRESUMO
5'-Phosphoribosylpyrophosphate amidotransferase, which catalyzes the synthesis of phosphoribosylamine in the de novo synthesis of purine nucleotides, has been detected and partially purified approx. 800-fold from Artemia sp. nauplii. The apparent Km values for 5'-phosphoribosyl 1-pyrophosphate as substrate were 0.7 mM and 0.4 mM in the presence of glutamine and ammonia as nitrogenous sources, respectively, and the enzymatic activity was inhibited by purine 5'-ribonucleotide compounds and 5', 5'''-p1, p4-diguanosine tetraphosphate.
Assuntos
Amidofosforribosiltransferase/metabolismo , Artemia/enzimologia , Pentosiltransferases/metabolismo , Amidofosforribosiltransferase/antagonistas & inibidores , Amidofosforribosiltransferase/isolamento & purificação , Amônia/farmacologia , Animais , Artemia/crescimento & desenvolvimento , Cromatografia , Glutamina/farmacologia , Cinética , Fosforribosil Pirofosfato/metabolismo , Nucleotídeos de Purina/farmacologiaAssuntos
Amidofosforribosiltransferase/isolamento & purificação , Fígado/enzimologia , Pentosiltransferases/isolamento & purificação , Amidofosforribosiltransferase/análise , Amidofosforribosiltransferase/imunologia , Animais , Reações Cruzadas , Masculino , Oxigênio/farmacologia , Fenantrolinas/farmacologia , Ratos , Ratos EndogâmicosAssuntos
Amidofosforribosiltransferase/metabolismo , Glucagon/farmacologia , Pentosiltransferases/metabolismo , Purinas/metabolismo , Amidofosforribosiltransferase/isolamento & purificação , Animais , AMP Cíclico/análise , Relação Dose-Resposta a Droga , Fígado/enzimologia , Masculino , Fosforribosil Pirofosfato/análise , Fosforilação , Proteínas Quinases/farmacologia , Ratos , Ratos Endogâmicos , Fatores de TempoAssuntos
Amidofosforribosiltransferase/metabolismo , Escherichia coli/enzimologia , Glutamatos/análise , Pentosefosfatos/análise , Pentosiltransferases/metabolismo , Fosforribosil Pirofosfato/análise , Ribosemonofosfatos/análise , Amidofosforribosiltransferase/isolamento & purificação , Sequência de Aminoácidos , Amônia/metabolismo , Sítios de Ligação , Radioisótopos de Carbono , Cromatografia por Troca Iônica/métodos , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico , Glutamina/metabolismo , Cinética , Técnica de Diluição de Radioisótopos , Espectrofotometria Ultravioleta/métodosRESUMO
Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de novo purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent Mr of 8 X 10(6), but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required Mg2+, and PRPPMg3- was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the Vm with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine, PPi, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.
Assuntos
Amidofosforribosiltransferase/metabolismo , Pentosiltransferases/metabolismo , Plantas/enzimologia , Amidofosforribosiltransferase/isolamento & purificação , Amônia/farmacologia , Glutamina/metabolismo , Inosina Monofosfato/farmacologia , Cinética , Peso Molecular , Glycine maxAssuntos
Amidofosforribosiltransferase/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida , Ligases/metabolismo , Linfócitos/metabolismo , Pentosiltransferases/metabolismo , Purinas/biossíntese , Amidofosforribosiltransferase/isolamento & purificação , Humanos , Cinética , Ligases/isolamento & purificação , Ativação LinfocitáriaRESUMO
Human and other mammalian forms of ATase, including the Chinese hamster enzyme, are oxygen-sensitive enzymes and human ATase, like the enzyme from B. subtilis, is an iron-sulfur protein. When protein synthesis is inhibited in cultured Chinese hamster cells, ATase activity is lost in an oxygen-dependent reaction. The hypothesis is developed that the sensitivity of ATase to oxygen inactivation controls the rate of degradation of this enzyme in mammalian cells, similar to the mechanism which has been demonstrated for regulation of ATase degradation in B. subtilis.
Assuntos
Amidofosforribosiltransferase/antagonistas & inibidores , Oxigênio/metabolismo , Pentosiltransferases/antagonistas & inibidores , Amidofosforribosiltransferase/isolamento & purificação , Animais , Células Cultivadas , Cricetinae , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/enzimologia , Humanos , Ferro/análise , Cinética , Ligantes , Mesocricetus , Placenta/enzimologia , Gravidez , Sulfetos/análiseRESUMO
Glutamine 5-phosphoribosylamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was purified in large amounts from an Escherichia coli strain harboring a purF hybrid plasmid. Purified E. coli amidophosphoribosyltransferase lacks iron as well as other trace metals as determined by x-ray fluorescence spectrometry. The NH2-terminal amino acid sequence of the enzyme was determined and is in agreement with that deduced from the DNA sequence. [6-14C] Diazo-5-oxo-norleucine (DON), an active site-directed affinity analog of glutamine, selectively inactivated the glutamine-dependent amidophosphoribosyltransferase. Inactivation was accompanied by incorporation of 1 eq of [6-14C]DON per enzyme subunit. A 10-residue cyanogen bromide peptide labeled by [6-14C]DON was isolated and sequenced. The NH2-terminal cysteine of amidophosphoribosyltransferase was determined to be the residue alkylated by [6-14C]DON. These results establish that the NH2-terminal cysteine is the active site residue required for the glutamine amide transfer function of the enzyme. The experiments reported in this and the preceding article (Tso, J. Y., Zalkin, H., van Cleemput, M., Yanofsky, C., and Smith, J. M. (1982) 257, 3525-3531) demonstrate the application of affinity labeling, rapid peptide purification by high pressure liquid chromatography, and nucleotide sequence determination of a structural gene to localize an amino acid residue, peptide fragment, or functional domain in a long protein chain.
Assuntos
Amidofosforribosiltransferase/genética , Clonagem Molecular , Escherichia coli/enzimologia , Pentosiltransferases/genética , Oligoelementos/análise , Amidofosforribosiltransferase/isolamento & purificação , Amidofosforribosiltransferase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Genes , Glutamina/metabolismo , Fragmentos de Peptídeos/análise , Ligação ProteicaRESUMO
A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented.
Assuntos
Amidofosforribosiltransferase/isolamento & purificação , Bacillus subtilis/enzimologia , Pentosiltransferases/isolamento & purificação , Aminoácidos/análise , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Proteínas Ferro-Enxofre/análise , Substâncias Macromoleculares , Peso Molecular , EspectrofotometriaAssuntos
Amidofosforribosiltransferase/antagonistas & inibidores , Fígado/enzimologia , Pentosiltransferases/antagonistas & inibidores , Nucleotídeos de Purina/farmacologia , Nucleotídeos de Pirimidina/farmacologia , Amidofosforribosiltransferase/isolamento & purificação , Animais , Cátions Bivalentes , Fenômenos Químicos , Química , Galinhas , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de HidrogênioAssuntos
Amidofosforribosiltransferase/metabolismo , Pentosiltransferases/metabolismo , Amidofosforribosiltransferase/antagonistas & inibidores , Amidofosforribosiltransferase/isolamento & purificação , Amino Açúcares/biossíntese , Amônia/metabolismo , Animais , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Glutamina/metabolismo , Humanos , Cinética , Fígado/enzimologia , Camundongos , Peso Molecular , Fosforribosil Pirofosfato/metabolismo , Placenta/enzimologia , Nucleotídeos de Purina/metabolismo , Ratos , Ribosemonofosfatos/biossínteseRESUMO
A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.