Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis

We isolated pleiotropic mutants of Klebsiella aerogenes with the transposon Tn5 which were unable to utilize a variety of poor sources of nitrogen. The mutation responsible was shown to be in the asnB gene, one of two genes coding for an asparagine synthetase. Mutations in both asnA and asnB were necessary to produce an asparagine requirement. Assays which could distinguish the two asparagine synthetase activities were developed in strains missing a high-affinity asparaginase. The asnA and asnB genes coded for ammonia-dependent and glutamine-dependent asparagine synthetases, respectively. Asparagine repressed both enzymes. When growth was nitrogen limited, the level of the ammonia-dependent enzyme was low and that of the glutamine-dependent enzyme was high. The reverse was true in a nitrogen-rich (ammonia-containing) medium. Furthermore, mutations in the glnG protein, a regulatory component of the nitrogen assimilatory system, increased the level of the ammonia-dependent enzyme. The glutamine-dependent asparagine synthetase was purified to 95%. It was a tetramer with four equal 57,000-dalton subunits and catalyzed the stoichiometric generation of asparagine, AMP, and inorganic pyrophosphate from aspartate, ATP, and glutamine. High levels of ammonium chloride (50 mM) could replace glutamine. The purified enzyme exhibited a substrate-independent glutaminase activity which was probably an artifact of purification. The tetramer could be dissociated; the monomer possessed the high ammonia-dependent activity and the glutaminase activity, but not the glutamine-dependent activity. In contrast, the purified ammonia-dependent asparagine synthetase, about 40% pure, had a molecular weight of 80,000 and is probably a dimer of identical subunits. Asparagine inhibited both enzymes. Kinetic constants and the effect of pH, substrate, and product analogs were determined. The regulation and biochemistry of the asparagine synthetases prove the hypothesis strongly suggested by the genetic and physiological evidence that a glutamine-dependent enzyme is essential for asparagine synthesis when the nitrogen source is growth rate limiting.     

The topology of the glutamine and ATP binding sites of human asparagine synthetase.

Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.     

Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies

Thirteen stable hybridoma cell lines producing monoclonal antibodies specific for asparagine synthetase were established and one monoclonal antibody was chosen to produce an immunoaffinity resin for the purification of asparagine synthetase. Bovine pancreatic asparagine synthetase was purified to a specific activity of 395 nmol of Asn produced/min/mg. Electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 54,000 polypeptide. Prior cross-linking with dimethyl suberimidate resulted in a band at Mr = 52,500 (monomer) and two additional bands at Mr = 97,000 and 98,000 (dimers), suggesting the possibility of a heterogeneous enzyme population with slight differences in subunit composition. The ratio of Gln-dependent and NH3-dependent asparagine synthetase activities was constant for immunoaffinity-purified enzyme, but the ratios of glutaminase activity to synthetase activities varied, suggesting separate aspartate and glutamine binding sites. The monoclonal antibodies were tested as inhibitors of the Gln-dependent and NH3-dependent asparagine synthetase activities as well as for inhibition of the glutaminase activity of the enzyme. Two antibodies inhibited Gln- and NH3-dependent synthesis of asparagine, but did not affect the glutaminase activity of immunoaffinity-purified asparagine synthetase. A third monoclonal antibody inhibited Gln-dependent synthesis of asparagine and glutaminase activity, but activated NH3-dependent asparagine synthetase activity. These data are discussed in terms of multiple substrate binding domains within the asparagine synthetase molecule.     

The N-terminal cysteine of human asparagine synthetase is essential for glutamine-dependent activity

Site-specific mutagenesis was used to replace the N-terminal cysteine in human asparagine synthetase by an alanine. The mutant enzyme was expressed in the yeast Saccharomyces cerevisiae, and the asparagine synthetase activity was analyzed in vitro. The mutation resulted in the loss of the glutamine-dependent asparagine synthetase activity, while the ammonia-dependent activity remained unaffected. These results confirm the existence of a glutamine amidotransfer domain with an N-terminal cysteine essential for the glutamine-dependent asparagine synthetase activity.     

Kinetic mechanism of beef pancreatic L-asparagine synthetase

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.     

A convenient gHMQC-based NMR assay for investigating ammonia channeling in glutamine-dependent amidotransferases: studies of Escherichia coli asparagine synthetase B

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover. These observations are consistent with a model in which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependent asparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.     

Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase

Brzozowski, Thomas H. (Stanford University School of Medicine, Palo Alto, Calif.), and Sumner M. Kalman. Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase. J. Bacteriol. 91:2286-2290. 1966.-Earlier studies have shown that the carbamyl phosphate synthesis from ammonia in cell extracts of wild-type Escherichia coli is due to at least two enzymes, acetokinase and the glutamine-dependent carbamyl phosphate synthetase. Partial purification of the glutamine-dependent carbamyl phosphate synthetase and acetokinase fails to separate from these enzymes this ammonia-dependent activity. An antibody to the partially purified acetokinase was prepared and used to determine the distribution of the ammonia-dependent activity in wild-type organisms and single-step arginine-uracil-requiring mutants with respect to the two enzymes. Such a study was possible because the antibody inhibits acetokinase but not the glutamine-utilizing carbamyl phosphate synthetase. Enzyme inhibition obtained by the stepwise addition of the antibody to cell extracts indicates that all of the ammonia-dependent carbamyl phosphate synthesis observed in the arginine-uracil-requiring mutants is due to a protein in the acetokinase fraction, presumably acetokinase itself, since acetyl phosphate and carbamyl phosphate synthesis were inhibited in a parallel fashion. In wild-type organisms, there is only partial inhibition of the ammonia-dependent activity, even when enough antibody is added to produce maximal inhibition of acetokinase. It is suggested that this residue is due to the glutamine-dependent carbamyl phosphate synthetase, for the ratio of the antibody insensitive to antibody sensitive ammonia-dependent activity present in cell extracts of the two wild-type organisms reported is qualitatively proportional to the level of carbamyl phosphate synthetase present relative to acetokinase.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

Anthranilate synthase of Neurospora crassa: reaction and labeling with glutamine analogs

The multifunctional enzyme complex, anthranilate synthase from Neurospora crassa, irreversibly loses its glutamine-dependent anthranilate synthase activity on exposure to the reactive glutamine analogs DON and azaserine. Inactivation depends on the presence of the substrate chorismate, is enhanced by the cofactor Mg⁺², and is antagonized by glutamine. Inactivation correlates well with the incorporation of [¹⁴C]DON into the protein with modification localized to the β subunit (M_r 84,000) of the complex, demonstrating directly that the β subunit provides the glutamine binding site for the glutamine-dependent anthranilate synthase reaction. The slower and less extensive loss of ammonia-dependent anthranilate synthase activity indicates that maximum expression of the ammonia-dependent anthranilate synthase activity by the α subunit also depends on the interaction with an active glutamine amidotransferase domain of the β subunit.     

Purification and characterization of beef pancreatic asparagine synthetase

Bovine pancreatic asparagine synthetase has been partially purified using ammonium sulfate fractionation, DEAE ion-exchange, Cibacron Blue affinity chromatography, and HPLC anion-exchange chromatography to a specific activity of 170 nmol asparagine produced min-1 mg protein-1, or 1400-fold, from a crude homogenate. Using HPLC size exclusion chromatography, an apparent molecular weight of 110,000-120,000 was determined. An aspartyl-adenylate intermediate was found to occur by demonstrating an 18O transfer from [18O]Asp to AMP that was detected with 31P NMR. A number of divalent metals were found to be able to replace magnesium with retention of activity, but none produced as high an activity as Mg2+, and the stoichiometry of the ATP/Mg2+ ratio was found to be 1. The chloride ion was found to stimulate the glutamine-dependent and glutaminase reactions, but the ammonia-dependent reaction was inhibited. Chloride appeared to be a competitive inhibitor with respect to ammonia and produced negative cooperativity.     

Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.     

Studies of the ammonia-dependent reaction of beef pancreatic asparagine synthetase

We have studied the asparagine synthetase reaction with regard to the ammonia-dependent production of asparagine. Hydroxylamine was shown to be an alternate substrate for the asparagine synthetase reaction, and some of its kinetic properties were examined. The ammonia-dependent reaction was examined with regard to inhibition by asparagine. It was found that asparagine inhibition was partial competitive with respect to ammonia, regardless of the concentration of aspartate. However, when MgATP was not saturating, the inhibition by asparagine became linear competitive. These results were interpreted to be consistent with a kinetic mechanism for asparagine synthetase where ammonia is bound to the enzyme followed by MgATP causing asparagine release.     

Topographical separation of the catalytic sites of asparagine synthetase explored with monoclonal antibodies

Monoclonal antibodies which inhibited the enzymatic activity of bovine pancreatic asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. Three antibodies which all inhibited glutamine- and NH3-dependent synthesis of asparagine bound to a common antigenic region. A fourth monoclonal antibody which interfered with glutamine binding or cleavage but not with NH3-dependent synthesis of asparagine was mapped to a separate region of the enzyme surface. These findings suggest a topographical separation between the aspartyl-AMP and glutamine-binding sites of bovine pancreatic asparagine synthetase. Three noninhibitory antibodies exhibited conformation-dependent binding and were mapped to a third region of the enzyme. Binding assays were used to demonstrate extensive cross-reaction of these antibodies with asparagine synthetases isolated from bovine liver and sheep pancreas. Substantial cross-reactions were also seen with the enzyme isolated from rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Of the four antibodies that inhibited glutamine- and NH3-dependent synthesis of asparagine from ruminant species, only one bound to and inhibited the enzyme from rat liver or mouse cells, which suggests significant structural differences between the ruminant and rodent enzymes.     

Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas

Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.     

The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln

The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4 s(-1)/mM and 3059.3 s(-1)/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys(52), Ser(128), Ser(152)) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA(Asn) and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.     

Kinetic mechanism of asparagine synthetase from Vibrio cholerae

Asparagine synthetase B (AsnB) catalyzes the formation of asparagine in an ATP-dependent reaction using glutamine or ammonia as a nitrogen source. To obtain a better understanding of the catalytic mechanism of this enzyme, we report the cloning, expression, and kinetic analysis of the glutamine- and ammonia-dependent activities of AsnB from Vibrio cholerae. Initial velocity, product inhibition, and dead-end inhibition studies were utilized in the construction of a model for the kinetic mechanism of the ammonia- and glutamine-dependent activities. The reaction sequence begins with the ordered addition of ATP and aspartate. Pyrophosphate is released, followed by the addition of ammonia and the release of asparagine and AMP. Glutamine is simultaneously hydrolyzed at a second site and the ammonia intermediate diffuses through an interdomain protein tunnel from the site of production to the site of utilization. The data were also consistent with the dead-end binding of asparagine to the glutamine binding site and PP(i) with free enzyme. The rate of hydrolysis of glutamine is largely independent of the activation of aspartate and thus the reaction rates at the two active sites are essentially uncoupled from one another.     

Anion regulation of lupin asparagine synthetase: Chloride activation of the glutamine-utilizing reactions

Small monovalent anions strongly activate glutamine-dependent asparagine synthesis and glutamine hydrolysis catalysed by highly purified asparagine synthetase (EC 6.3.5.4) from cotyledons of Lupinus luteus seedlings. Cl^? and Br^? are most effective, but F^?, I^?, NO₃^? and CN^? also stimulate both reactions. The synthetase reactions with NH₃, or NH₂OH are only slightly stimulated by Cl^? and Br^?, indicating that the anions selectively accelerate the reactions involving glutamine cleavage. In asparagine synthesis Cl^? is a competitive activator vs glutamine and a noncompetitive activator vs MGATP and aspartate. Addition of Cl^? changes the substrate saturation kinetics of glutamine from negatively cooperative to normal hyperbolic and causes a 50-fold increase in the affinity for glutamine. The inherent glutaminase activity of the enzyme is enhanced up to 30-fold by addition of Cl^?, MgATP and aspartate. Thus, ligands of the synthetase reaction act as allosteric activators of the glutaminase step in the enzyme mechanism.     

Eukaryotic NAD+ synthetase Qns1 contains an essential,obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase

NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.     

Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Sch?n, A., Kannangara, C. G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an Mr of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent Mr of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an alpha 2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.