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PubMed1981

[Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates].

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.

Adenosine Triphosphate↗
PubMed1981

Isoleucyl-tRNA synthetase from Baker's yeast. Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate.

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Adenosine Triphosphate↗
PubMed1983

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion.

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.

Clostridium↗
PubMed2005

Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements.

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.

Adenosine Triphosphate↗
PubMed1980

S-Adenosylmethionine synthetase from Escherichia coli.

Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.

Adenylyl Imidodiphosphate↗
PubMed1976

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.

Adenine Nucleotides↗
PubMed2005

The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.

The high energy sulfate donor 3'-phosphoadenosine-5-phosphosulfate (PAPS) is used for sulfate conjugation of extracellular matrix, hormones and drugs. Human PAPS synthetase 1 catalyzes two subsequent reactions starting from ATP and sulfate. First the ATP sulfurylase domain forms APS, then the APS kinase domain phosphorylates the APS intermediate to PAPS. Up to now the interaction between the two enzymatic activities remained elusive, mainly because of missing structural information. Here we present the crystal structure of human PAPSS1 at 1.8 angstroms resolution. The structure reveals a homodimeric, asymmetric complex with the shape of a chair. The two kinase domains adopt different conformational states, with only one being able to bind its two substrates. The asymmetric binding of ADP to the APS kinase is not only observed in the crystal structure, but can also be detected in solution, using an enzymatic assay. These observations strongly indicate structural changes during the reaction cycle. Furthermore crystals soaked with ADP and APS could be prepared and the corresponding structures could be solved.

Adenosine Diphosphate↗
PubMed1975

The catalytic mechanism of amino acid:tRNA ligases. Synergism and formation of the ternary enzyme-amino acid-ATP complex.

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.

Adenosine↗
PubMed2002

A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactivations that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differential responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.

Adenosine Triphosphate↗
PubMed1989

Molecular cloning and expression in Escherichia coli of the Bacillus licheniformis bacitracin synthetase 2 gene.

The structural genes for the entire bacitracin synthetase 2 (component II) and for a part of the putative bacitracin synthetase 3 (component III) from Bacillus licheniformis ATCC 10716 were cloned and expressed in Escherichia coli. A cosmid library of B. licheniformis DNA was constructed. The library was screened for the ability to produce bacitracin synthetase by in situ immunoassay using anti-bacitracin synthetase antiserum. A positive clone designated B-15, which has a recombinant plasmid carrying about a 32-kilobase insert of B. licheniformis DNA, was further characterized. Analysis of crude cell extract from B-15 by polyacrylamide gel electrophoresis and Western blotting (immunoblotting) showed that the extract contains two immunoreactant proteins with high molecular weight. One band with a molecular weight of about 240,000 comigrates with bacitracin synthetase 2; the other band is a protein with a molecular weight of about 300,000. Partial purification of the gene products encoded by the recombinant plasmid by gel filtration and hydroxyapatite column chromatography revealed that one gene product catalyzes L-lysine- and L-ornithine-dependent ATP-PPi exchange reactions which are characteristic of bacitracin synthetase 2, and the other product catalyzes L-isoleucine-, L-leucine, L-valine-, and L-histidine-dependent ATP-PPi exchange activities, suggesting the activities of a part of bacitracin synthetase 3. Subcloning experiments indicated that the structural gene for bacitracin synthetase 2 is located near the middle of the insert.

Bacillus↗
PubMed1985

Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.

Amino Acid Isomerases↗
PubMed1987

Catabolic enzymes of the acetogen Butyribacterium methylotrophicum grown on single-carbon substrates.

When grown on formate, formate-CO, and methanol-CO, Butyribacterium methylotrophicum contained high levels of tetrahydrofolate (H4folate) and required enzymes, carbon monoxide dehydrogenase, formate dehydrogenase, and hydrogenase. The activities of methylene-H4folate reductase were comparable to other H4 folate activities (which ranged from 0.55 to 9.28 mumol/min per mg of protein) when measured by an improved procedure. The H4folate activities in formate-grown cells were twice those found in formate-CO-grown cells. This result correlated with a growth yield on formate that was one-half that on formate-CO. The stoichiometry of the formyl-H4folate synthetase reaction was 1 mol of ATP per 1 mol of formate. The methylene-H4folate dehydrogenase was NAD+ dependent. We conclude that B. methylotrophicum utilizes these enzymes in homoacetogenic catabolism.

Aldehyde Oxidoreductases↗
PubMed1986

Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase.

New methods for the estimation of red cell gamma-glutamylcysteine synthetase and glutathione synthetase have been developed. gamma-32P ATP is allowed to equilibrate until the gamma and beta phosphate groups are equally labelled. The amount of 32Pi released in the presence of glutamic acid and cysteine, the substrates for GC-S or in the presence of gamma-glutamylcysteine and glycine, the substrates of GSH-S, is measured. This is accomplished by extraction of the phosphomolybdate complex into isobutanol-benzene. The methods are linear with time and hemolysate concentration. Normal values are presented.

Adenosine Triphosphate↗
PubMed1985

Effect of amoscanate derivative CGP 8065 on aminoacyl-tRNA synthetases in Ascaris suum.

Evidence is provided for the occurrence of a multienzyme complex consisting of several aminoacyl-tRNA synthetases besides 'free enzymes' in Ascaris suum. The molecular mass of this complex was calculated to be about 10(6) daltons, compared to about 150 000 daltons for the seryl-tRNA synthetase. Leucyl-, isoleucyl-, arginyl- and lysyl-tRNA synthestases were found in the high molecular weight fraction. The Michaelis constants of these aminoacyl-tRNA synthetases were found to be in the range of 4 to 10 microM for amino acids and of 0.1 to 1.0 mM for ATP. Leucyl- and isoleucyl-tRNA synthetase interact with the amoscanate-derivative CGP 8065. The inhibition constants were determined to be 34 microM and 8 microM, respectively. The type of inhibition was found to be competitive with respect to ATP. It is proposed that the interference of CGP 8065 with the charging of tRNA might be another target for the chemotherapeutic attack of this amoscanate derivative.

Amino Acyl-tRNA Synthetases↗
PubMed2005

Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain.

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.

Adenosine Monophosphate↗
PubMed1998

Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

Aerobiosis↗
PubMed1996

Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials.

B16-F10 and B16-BL6 are B16 mouse melanoma sublines that preferentially metastasize to the lung following i.v. and s.c. injections, respectively. To study molecular mechanisms underlying the different metastatic behaviors exhibited by the B16 melanoma sublines, we performed differential hybridization of the genes transcribed in these cells and compared their expression levels. We isolated four genes that were highly expressed in B16-F10 cells but not in B16-BL6 cells: TI-225 (polyubiquitin), TI-229 (pyruvate kinase), TI-241 (LRF-1 homologue), and TI-227 (novel gene). Triosephosphate isomerase, 10-formyltetrahydrofolate dehydrogenase, tyrosinase-related protein 2, cytochrome c oxidase, ATP synthetase alpha subunit, RNA helicase, and ribosomal protein (L37, J1, acidic phosphoprotein), however, showed higher expression in B16-BL6 cells than in B16-F10 cells. Among these clones, transfection of TI-241 into the low metastatic clone F1 converted the parental cells from low- into high-metastatic cells. TI-241 may regulate the expression of various genes as a transcription factor in the complex process of metastasis.

Amino Acid Sequence↗
PubMed1977

Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid.

The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases. Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme. In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex. With all five ligases the steady state rate of transfer from the preformed aminoacyl-adenylate complex to tRNA was approximately the same as that of the overall reaction.

Adenosine Monophosphate↗