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PubMed1979

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.

Adenosine Triphosphate↗
PubMed2001

The structure of the PII-ATP complex.

PII is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of PII was solved using a hexagonal crystal form (form I). The more physiologically relevant form of PII is a complex with small molecule effectors. We describe the structure of PII with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of PII with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the PII/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that PII and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.

Adenosine Triphosphate↗
PubMed1990

Firefly luciferase synthesizes P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates.

The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275-8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959-2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E-aminoacyl-AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E-luciferin-AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl-containing intermediates and releasing PPi may produce dinucleoside polyphosphates.

Dinucleoside Phosphates↗
PubMed1982

Comparison of the hydrolysis patterns of several tRNAs by cobra venom ribonuclease in different steps of the aminoacylation reaction.

The hydrolysis of several tRNAs by an endonuclease extracted from the venom of Naja oxiana and specific for double-stranded, or at least highly ordered, regions has been studied under various experimental conditions. It is shown that the hydrolysis patterns of yeast tRNAPhe, tRNAVal and tRNAAsp in the isolated state are similar, most of the cuts occurring in the anticodon and acceptor stems. Ionic conditions are able to modify the hydrolysis pattern. The origin of these modifications is discussed. The protection against ribonuclease action, afforded to tRNAPhe, tRNAVal and tRNAAsp by the cognate aminoacyl-tRNA synthetase, is analyzed. It is shown that in all cases the anticodon stem is protected. The 3'-terminal region does not seem to be tightly engaged in the complex with the aminoacyl-tRNA synthetase. These results are discussed in the light of information on contact areas previously obtained by ultraviolet cross-linking techniques. The effects of the small ligands (ATP and amino acid) on the protection afforded to the tRNA by the cognate synthetase, have been studied. In the valine and aspartic acid systems, ATP induced a modification of the tRNA-enzyme complex leading to differences in the hydrolysis pattern of the 3'-accepting region. The effects of aminoacylation on the cleavage of tRNAPhe, tRNAVal and tRNAAsp were also studied. Whereas no modification of the cleavage map was observed in the aspartic system, aminoacylation resulted in slight but significant modifications of the hydrolysis pattern for tRNAPhe and tRNaVal in the 3'-terminal region.

Adenosine Triphosphate↗
PubMed1983

Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent.

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1995

Analysis of the role of the KMSKS loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles.

A mobile loop in tyrosyl-tRNA synthetase, which corresponds to the KMSKS signature sequence of class I aminoacyl-tRNA synthetases, destabilizes the E.Tyr.ATP complex but stabilizes the following E.[Tyr-ATP]not equal to transition state for the formation of E.Tyr-AMP. Three amino acid residues in the mobile loop, K230, K233, and T234, are known to be primarily responsible for these effects. We now analyze the network of interactions between these three amino acids using multiple mutant free energy cycles. The complete characterization of the coupling energies within the mobile loop allows each of the steps leading to the formation of the transition state complex to be dissected into its energetic components. In particular, it is found that, in the absence of a functional mobile loop, there is synergistic coupling between the tyrosine and ATP substrates (i.e., each enhances the binding affinity of the other) which stabilizes the E.Tyr.ATP intermediate preceding the transition state complex. Thus, the mobile loop disrupts the synergism between the ATP and tyrosine substrates, using the ATP binding energy to stabilize the transition state for the reaction. Whereas the net effect of the mobile loop in the E.Tyr.ATP complex results from several conflicting side chain interactions that tend to offset each other, conflicting interactions in the E.[Tyr-ATP]not equal to transition state complex have been minimized and stabilizing pairwise interactions between the K230, K233, and T234 side chains are optimized. The tight coupling between the side chains of K230, K233, and T234 suggests that the mobile loop adopts a highly constrained conformation during formation of the transition state complex. These results quantitatively demonstrate the importance of side chain interactions in enzyme catalysis and illustrate the use of binding energy to stabilize the transition state of a reaction and the presence of unfavorable interactions to destabilize the ground state.

Adenosine Triphosphate↗
PubMed1986

Substrate specificity of formylglycinamidine synthetase.

Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. [Buchanan, J. M., Ohnoki, S., & Hong, B. S. (1978) Methods Enzymol. 51, 193-201] (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH2NHCHO) has been replaced by R = CH3, CH2OH, CH2Cl, CH2NH3, CH2NHCOCH3, CH2NHCOCH2Cl, CH2NHCO2CH2Ph, and L-CHC-H3NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH3, CH2OH, and CH2NHCOCH3 and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH2NHCOCH3 causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH2NHCHO) and the FGAR analogue (R = CH2NHCHOCH3) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. [18O]-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.

Adenosine Triphosphatases↗
PubMed2001

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis.

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

Acyl Carrier Protein↗
PubMed2004

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains.

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.

Adenosine Phosphosulfate↗
PubMed1982

Rat-liver fatty-acid-synthesizing complex.

Under conditions favoring lipogenesis, a high-molecular-weight species of acetyl-CoA carboxylase was isolated that did not co-sediment with the in vitro polymerized enzyme. Assays for ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase indicated that all three enzymes were associated together as a high-molecular-weight complex and that under low-lipogenic conditions the level of these enzymes decreased. Phosphorylation of the isolated complex shifted it toward a lower molecular weight.

ATP Citrate (pro-S)-Lyase↗
PubMed1995

Electrostatic potential in aminoacylation by aspartyl-tRNAs synthetase.

Based upon the X-ray structures of complexes between tRNAAsp and aspRS including ATP or Asp-AMP, several electrostatic potentials were calculated by solving the Poisson-Boltzmann equation. The potentials indicate clearly that a Mg2+ ion is essential for binding of ATP and that aspartate is identified electrostatically. The alpha-carboxyl group is forced to contact with the alpha-phosphorus atom of ATP, suggesting its inversion to form an Asp-AMP. When the cognate tRNA is bound to the aspRS:Asp-AMP complex, the 3'-hydroxyl group is located in an electrostatically favorable position to transfer the amino acid as a class II aminoacylation.

Acylation↗
PubMed1988

[Enzyme systems of the substrate and cofactor supply of hyperlipogenesis in non-insulin-dependent diabetes].

Enzymatic systems of hepatic hyperlipogenesis supply by substrate (acetyl-CoA) and cofactors (NADPH and ATP) were studied in experiments on diabetic C57Bl/Ks J mice (db/db) that served as a model of non-insulin dependent diabetes. The rise in acetyl-CoA synthetase activity catalyzing the primary step of lipogenesis from acetate has been found, while pyruvate dehydrogenase complex activity did not differ from the control and ATP-citrate lyase activity was lowered. Hyperlipogenesis in non-insulin dependent diabetes was induced by the activation of cellular energy supply revealed in enhanced 2-oxoglutarate dehydrogenase activity and elevated ATP level, as well as changes in the activity ratio of NADPH supply and utilization and the rise in fructose-1,6-diphosphate, allosteric effector of fatty acid synthetase, which resulted in the increase of the enzyme activity and created wider potentials of NADPH utilization as a reducing equivalent in lipogenesis.

ATP Citrate (pro-S)-Lyase↗
PubMed1993

On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes.

For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions. Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T. (1992) Biochemistry 31, 3955-3963). In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins. When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline. The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent. It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate. If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly.

Bacterial Proteins↗
PubMed1987

The mechanism of cytochrome oxidase and other reaction centres for electron/proton pumping.

The functional significance of the metal centres of cytochrome oxidase is deduced from the ways in which the centres are bound into its peptides. To this end use is made of structural knowledge of other metalloproteins for dioxygen binding, haemocyanin and haemoglobin, and for electron transfer, cytochromes b and azurin. The order and manner in which the motions of helical sections of the oxidase are linked to proton pumping are suggested and a comparison is made with other proton pumps, for example that of ATP synthetases.

Animals↗
PubMed1979

Kinetics of the D and I glycogen synthetase forms and their interconversion, in the sea scallop Pecten maximus.

In extracts from the adductor muscle of the shell-fish, Pecten maximus, glycogen synthetase (EC.2.4.1.11) was found. The enzyme occurs predominantly as D form (glucose-6-P dependent for activity). An I form (G-6-P independent) was also present. Kinetics of glycogen synthetase showed that the Ka for G-6-P in the D form was 10 fold higher than in the I form. Both forms of glycogen synthetase were interconverted through reactions catalyzed by phosphatase and kinase enzymes respectively. Glucose-6-P and Mg+2 must be present to stabilize glycogen synthetase and to activate the synthetase D phosphatase, found in the 90,000 X g protein-glycogen complex. The conversion of synthetase D to I was inhibited by F-, glycogen, ATP and UTP. When F- was present the effect of G-6-P on synthetase and phosphatase suggested that conversion involved the existence of more than a single glycogen synthetase phosphatase enzyme. ATP and Mg+2 were necessary for the conversion of synthetase I to D, and the conversion was stimulated by cAMP.

Cyclic AMP↗
PubMed1984

Crystallization of substrate and product analog complexes of tryptophanyl-tRNA synthetase.

We have prepared crystals of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus complexed to tryptophan (type II*), and to tryptophanyl-3'(2')-ATP (type IV). The latter compound is a product analog, enzymatically synthesized by acyl transfer of tryptophan from the tryptophanyl-5'-AMP intermediate to a second molecule of ATP. It resembles the 3'-terminal fragment, tryptophanyl-3'(2')-adenosine, of Trp-tRNATrp. Both crystal forms diffract to high resolution. Although both forms are grown from 2 M K2HPO4, they are dramatically different in the shape of the unit cell and in space group symmetry. Type II* crystals are monoclinic (space group P21). However, low-resolution reflections obey the symmetry of space group P321, which indicates both the existence and the location of noncrystallographic symmetry in the monoclinic unit cell. Type IV crystals belong to space group P41212 (or its enantiomorph) and the unit cell is elongated along the fourfold screw axis. Analysis of molecular packing suggests that intermolecular contacts in the two crystal types are very different. Thus, the two structures may exhibit conformational differences related to catalysis by this enzyme. Solution of type II* and type IV crystal structures may provide representations resembling a Michaelis complex and an acyl transfer product complex.

Adenosine Triphosphate↗
PubMed2002

Crystal structure of argininosuccinate synthetase from Thermus thermophilus HB8. Structural basis for the catalytic action.

Argininosuccinate synthetase catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its free form, complexed with intact ATP, and complexed with an ATP analogue (adenylyl imidodiphosphate) and substrate analogues (arginine and succinate) have been determined at 2.3-, 2.3-, and 1.95-A resolution, respectively. The structure is essentially the same as that of the Escherichia coli argininosuccinate synthetase. The small domain has the same fold as that of a new family of "N-type" ATP pyrophosphatases with the P-loop specific for the pyrophosphate of ATP. However, the enzyme shows the P-loop specific for the gamma-phosphate of ATP. The structure of the complex form is quite similar to that of the native one, indicating that no conformational change occurs upon the binding of ATP and the substrate analogues. ATP and the substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometrical orientation favorable to the catalytic action. The reaction mechanism so far proposed seems to be consistent with the locations of ATP and the substrate analogues. The reaction may proceed without the large conformational change of the enzyme proposed for the catalytic process.

Adenosine Triphosphate↗