Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ATP Synthetase Complexes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed1994

Refolding and release of tubulins by a functional immobilized groEL column.

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.

Adenosine Triphosphate↗
PubMed1999

Active site titration of gramicidin S synthetase 2: evidence for misactivation and editing in non-ribosomal peptide biosynthesis.

The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP(i) generation using active site titration measurements with [gamma-(32)P]ATP. The initial 'burst' of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non-cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.

Adenosine Triphosphate↗
PubMed1998

The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

Ammonia↗
PubMed2001

The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.

BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.

Adenosine Triphosphate↗
PubMed2006

Priming type II polyketide synthases via a type II nonribosomal peptide synthetase mechanism.

Benzoic acid priming of the enterocin and actinorhodin type II polyketide synthase complexes was accomplished in vitro via an unprecedented type II nonribosomal peptide synthetase-like mechanism involving the benzoate:acyl carrier protein (ACP) ligase EncN and the ACP EncC. The transfer of the aryl acid to the ACP is ATP-dependent, yet coenzyme A-independent, as characterized with radiolabeled substrates and protein mass spectrometry. Subsequent transport of the ACP-bound aryl group to the native enterocin and the aberrant actinorhodin ketosynthase chain length factor heterodimers was further demonstrated, thereby demonstrating the potential of this biocatalyst for engineering diverse aryl-primed aromatic polyketide agents.

Acyl Carrier Protein↗
PubMed1985

Regulation and properties of glutamine synthetase purified from Bacillus cereus.

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.

Ammonium Sulfate↗
PubMed1994

Quantitative analysis of crystal growth. Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis.

We show that quantitative analysis of replicated, full-factorial crystal growth experiments and, by implication, similar studies of a wide variety of other phenomena, can be a powerful tool for analyzing macromolecular systems with complex, interacting dependencies on functionally significant factors. Bacillus stearothermophilus tryptophanyl-tRNA synthetase crystallizes in three different crystal forms depending on the ligands present under otherwise identical conditions. Comparison of crystallographic space groups for complexes with different ligands reveals that the three forms entail at least two very different families of packing arrangements that are correlated with specific changes in the enzyme ligation state. One is associated with the ligand-free enzyme, substrate ligands, and the binding of the activated amino acid; the other results from the presence of high ATP concentrations and/or the synthesis of the unusual acyl-transfer product, tryptophanyl-2'(3') ATP. Together with previous physico-chemical studies of aminoacyl-tRNA synthetases, these observations suggest that the two families are related, respectively, to the biochemical processes of amino acid activation and acyl transfer. Further evidence that the crystal polymorphism results from an underlying protein conformational polymorphism has now been obtained by quantitative analysis of how crystal growth depends on pH and the substrates tryptophan and ATP. The analysis consists first in showing that crystallization conditions for the unliganded protein are very favorable, suggesting that variation in crystal growth induced by pH and substrates under otherwise identical conditions is due to their effects on the protein conformation and not on incidental perturbations of crystal growth, per se. Next, crystal growth experiments are shown to be reproducible enough to support statistical analysis of quantitative scores assigned to the results. Finally, the observed variation in scores can be attributed at high confidence levels chiefly to three effects: that of pH alone, the synergistic effects of pH plus tryptophan, and of tryptophan plus ATP. These statistical inferences are consistent with other biochemical data, and support the conclusions based on crystal packing that representative stages of the enzyme mechanism have been trapped in the different crystal forms. The pH-tryptophan interaction implies that there is a pH-dependent conformational change favoring high affinity substrate binding at high pH. The pH-ATP interaction implies that a subsequent conformational change, not previously considered, occurs between tryptophan activation and acyl transfer.

Adenosine Triphosphate↗
PubMed2006

Crystal structure of the pantothenate synthetase from Mycobacterium tuberculosis, snapshots of the enzyme in action.

Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, beta-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of beta-alanine. This structure confirms that binding of beta-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, and the reaction product AMP provide a step-by-step view of the PS-catalyzed reaction. A detailed reaction mechanism and its implications for inhibitor design are discussed.

Adenosine Monophosphate↗
PubMed2007

Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site.

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

Allosteric Site↗
PubMed1982

Essential arginine residue in gramicidin S synthetase 1 of Bacillus brevis.

Phenylalanine activation of gramicidin S synthetase 1 (GS 1) [EC 5.1.1.11] of Bacillus brevis is inhibited by phenylglyoxal. The inactivation of GS 1 by phenylglyoxal obeys pseudo-first-order kinetics and formation of a reversible enzyme-reagent complex prior to modification is indicated. Both ATP and phenylalanine prevent the inactivation by phenylglyoxal. ATP is competitive with phenylglyoxal, whereas phenylalanine is not. In the presence of ATP, one residue of arginine per mol of protein is protected from the modification as determined by amino acid analysis and incorporation of [7-14C]phenylglyoxal. These results indicate that a single arginine residue of GS 1 is essential for phenylalanine activation in binding the phosphate moiety of ATP.

Adenosine Triphosphate↗
PubMed1976

The kinetic mechanism of rat kidney gamma-glutamylcysteine synthetase.

A detailed kinetic investigation was made of the binding mechanism of gamma-glutamylcysteine synthetase purified from rat kidney. The results of initial rate and inhibition studies are consistent with a partially random mechanism in which ATP is the obligatory first substrate and both amino acids bind in a random order to the enzyme-ATP complex. Formation of the enzyme-substrate quaternary complex is necessary prior to release of products. This mechanism is consistent with previous binding studies with the enzyme and while it does not rule out participation of enzyme-bound gamma-glutamyl phosphate as an intermediate in catalysis, such an intermediate cannot be a discrete covalent complex.

Adenosine Diphosphate↗
PubMed1999

The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase.

The metabolite glutathione fulfills many important and chemically complex roles in protecting cellular components from the deleterious effects of toxic species. GSH combines with hydroxyl radical, peroxynitrite, and hydroperoxides, as well as reactive electrophiles, including activated phosphoramide mustard. This thiol-containing reductant also maintains so-called thiol-enzymes in their catalytically active form, and maintains vitamins C and E in their biologically active forms. The key step in glutathione synthesis, namely the ATP-dependent synthesis of gamma-glutamylcysteine, is the topic of this review. Details are presented on (a) the enzyme's purification and protein chemistry, (b) the successful cDNA cloning, and characterization of the genes responsible for the biosynthesis of this enzyme. After considering aspects of the role of overexpression of this synthetase in terms of cancer chemotherapy, attention is focused on post-translational regulation. The remainder of the review deals with the catalytic mechanism (including substrate specificity, reactions catalyzed, steady-state kinetics, and chemical mechanism) as well as the inhibition of the enzyme (via feedback inhibition, reaction with S-alkyl homocysteine sulfoximine inhibitors, the clinical use of buthionine sulfoximine with cancer patients, and inactivation by cystamine, chloroketones, and various nitric oxide donors).

Animals↗
PubMed1996

Overexpression of Bacillus subtilis phosphoribosylpyrophosphate synthetase and crystallization and preliminary X-ray characterization of the free enzyme and its substrate-effector complexes.

Bacillus subtilis phosphoribosylpyrophosphate (PRPP) synthetase has been expressed to high levels in an Escherichia coli host strain devoid of endogenous PRPP synthetase. A rapid and efficient purification protocol has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallization. Crystallization experiments, in combination with dynamic light scattering studies, have led to the production of three crystal forms of the enzyme. These forms include the free enzyme, the enzyme in a binary complex with the substrate adenosine triphosphate (ATP), and the enzyme in a quaternary complex with the substrate analog alpha, beta-methylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-5-P), and the allosteric inhibitor adenosine diphosphate (ADP). Diffraction data showed that all three crystal forms are suitable for structure determination. They crystallize in the same hexagonal space group, P6(3), with virtually identical unit cell dimensions of a = b = 115.6 angstroms, c = 107.8 angstrom, and with two molecules in the asymmetric unit. The self-rotation function showed the existence of a non-crystallographic twofold axis perpendicular to the c axis. The availability of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed.

Adenosine Triphosphate↗
PubMed2003

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold.

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.

Adenosine Diphosphate↗
PubMed2005

Kinetic measurement by LC/MS of gamma-glutamylcysteine ligase activity.

gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.

Amino Acid Sequence↗
PubMed1994

Interactions of nucleotides with fully unadenylylated glutamine synthetase from Salmonella typhimurium.

Glutamine synthetase (GS) catalyzes the ATP-dependent biosynthesis of glutamine from glutamate and ammonia in the presence of divalent cations. To gain insight into the structural basis of the feedback inhibition of GS by AMP, we have studied crystal structures of GS complexes with AMP and the related molecules: AMPPNP (a less hydrolyzable ATP analog), ADP, GDP, adenosine, and adenine. AMP is a feedback inhibitor of GS; ATP and ADP are cofactors, and AMPPNP, GDP, adenosine, and adenine are also GS inhibitors. GS used in this study is from Salmonella typhimurium and is free of covalent modification by adenylylation. All of the crystals examined contain two bound MN2+ ions per GS subunit. The X-ray structures show that all nucleotides bind at the same site, the cofactor ATP binding site, as do adenosine and adenine. Thus from X-ray structures, AMP, adenosine, adenine, and GDP would be expected to inhibit GS-Mn by competing with the substrate ATP for the active site. This suggestion from the crystal structures that AMP is competitive with respect to ATP is supported by kinetic measurements using the biosynthetic assay.

Adenine↗
PubMedDate not supplied

[Tryptophanyl-tRNA-synthetase: limited proteolysis by elastase and isolation of "one-site" enzyme].

Each subunit of the dimeric tryptophanyl-tRNA-synthetase from beef pancreas is subjected to limited hydrolysis by elastase in two stages, according to scheme: 60 00 +/- 2000 leads to 51 000 +/- 2000 leads to 40 000 +/- 1500 daltons. In the course of the second step tryptophanyl-tRNA-synthetase looses its enzymatic activity. In the presence of substrates the pattern of fragments does not change. Formation of tryptophanyladenylate enzyme complex decreases the rate of proteolysis. Using the ability of synthetase to form one mole of stable aminoacyladenylate per mole of synthetase, the "one-site" enzyme was obtained by action of elastase on aminoacyladenylate-enzyme complex. This "one-site" enzyme consists of two subunits, one of which has a molecular weight of 51 000 daltons and is active and the other has a molecular weight of 40 000 daltons and is inactive. The "one-site" enzyme had Km values for all substrates for both aminoacylation and ATP--[32P]PP exchange reactions which are similar to values of Km for the native enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase.

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.

Adenosine Monophosphate↗