Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis.

The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd(2+). The structure determined from X-ray diffraction data to 2.8-A resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP. Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation. In addition to the bound Cd(2+), the Cd(2+)-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site. Comparison of the Cd(2+)-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction. The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP. This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.     

Glutamine PRPP amidotransferase: snapshots of an enzyme in action

Recent studies of glutamine PRPP amidotransferase have provided a new understanding of the function and mechanism of this rather complicated enzyme that may be a paradigm for other complex enzymes. New insights have been gained into the mechanisms of catalysis in the active sites of the two half-reactions, catalytic coupling, allosteric control by feedback inhibitors and the channeling of reaction and metabolic intermediates.     

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.     

Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets

Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.     

Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis.

Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.     

Cloning and expression of avian glutamine phosphoribosylpyrophosphate amidotransferase. Conservation of a bacterial propeptide sequence supports a role for posttranslational processing

The cDNA for glutamine phosphoribosylpyrophosphate amidotransferase, the regulatory enzyme of de novo purine nucleotide biosynthesis, has been cloned for the first time from an animal. The derived amino acid sequence of the avian amidotransferase is homologous with amidotransferase sequences from bacteria and yeast. An 11-amino acid propeptide in Bacillus subtilis amidotransferase is conserved in the avian enzyme. Expression in Chinese hamster ovary (CHO) cells and Escherichia coli provides evidence for two post-translational maturation steps needed for synthesis of active enzyme: incorporation of an iron component and processing of the 11-amino acid propeptide. Functional complementation of a CHO amidotransferase mutant suggests that both maturation steps take place in CHO cells. In contrast, function in E. coli requires deletion of the sequence encoding the propeptide. Defective assembly of the iron component may restrict propeptide removal and activation of the avian amidotransferase in E. coli.     

Dual role for the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel. Interdomain signaling and intermediate channeling

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalyzes the first reaction of de novo purine nucleotide synthesis in two steps at two sites. Glutamine is hydrolyzed to glutamate plus NH(3) at an N-terminal glutaminase site, and NH(3) is transferred through a 20-A hydrophobic channel to a distal PRPP site for synthesis of phosphoribosylamine. Binding of PRPP is required to activate the glutaminase site (termed interdomain signaling) to prevent the wasteful hydrolysis of glutamine in the absence of phosphoribosylamine synthesis. Mutations were constructed to analyze the function of the NH(3) channel. In the wild type enzyme, NH(3) derived from glutamine hydrolysis was transferred to the PRPP site, and little or none was released. Replacement of Leu-415 at the PRPP end of the channel with an alanine resulted in a leaky channel and release of NH(3) to the solvent. Mutations in five amino acids that line the channel and two other residues required for the reorganization of phosphoribosyltransferase domain "flexible loop" that leads to formation of the channel perturbed channel function as well as interdomain signaling. The data emphasize the role of the NH(3) channel in coupling interdomain signaling and NH(3) transfer.     

Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.     

Xanthosine-5'-phosphate amidotransferase from Escherichia coli.

The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate. This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase. The so-called XMP aminase from the E. coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase. These data demonstrate that this enzyme from E. coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.     

Crystal structure of thymidylate synthase A from Bacillus subtilis

Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4-fold greater k(cat) than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 A resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in k(cat) found between the two enzymes.     

Topological chirality of iron-sulfur proteins

An examination of x-ray structures of single-cluster [4Fe-4S] proteins in the Protein Data Bank has revealed that all redox proteins and the glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase from Bacillus subtilis have a topological configuration arbitrarily designated as D, whereas the DNA repair enzyme endonuclease III from Escherichia coli has the opposite topological configuration, L. This is the first example in which both senses of topological chirality have been observed in a class of proteins. © 1997 John Wiley & Sons, Inc. Biopoly 42: 411–414, 1997     

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.     

Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases.

Carbamoyl phosphate synthetases (CPSs) utilize either glutamine or ammonia for the ATP-dependent generation of carbamoyl phosphate. In glutamine-utilizing CPSs (e.g. the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at a triad-type glutamine amidotransferase domain. Non-glutamine-utilizing CPSs (e.g. rat and human CPS I), lacking the catalytic cysteine residue, can generate carbamoyl phosphate only in the presence of free ammonia. Frog CPS I (fCPS I), unlike mammalian CPS Is, retains most of the glutamine amidotransferase residues conserved in glutamine-utilizing CPSs, including an intact catalytic triad, and could therefore be expected to use glutamine. Our work with native fCPS I provides the first demonstration of the inability of this enzyme to bind/utilize glutamine. To determine why fCPS I is unable to utilize glutamine, we compared sequences of glutamine-using and non-glutamine-using CPSs to identify residues that are present or conservatively substituted in all glutamine-utilizing CPSs but absent in fCPS I. We constructed the site-directed mutants Q273E, L270K, Q273E/N240S, and Q273E/L270K in E. coli CPS and have determined that simultaneous occurrence of the two substitutions, Gln-->Glu and Leu-->Lys, found in the frog CPS I glutamine amidotransferase domain are sufficient to eliminate glutamine utilization by the E. coli enzyme.     

Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 ? resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.     

Rat liver glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase [EC 2.4.2.14]. Purification and properties.

Glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (amidophosphoribosyltransferase), [EC 2.4.2.14] was purified 1,600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215,000, 200,000, and 195,000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density grandient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24 mM and 0.57 mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.     

A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro.

In the presence or absence of its regulatory factor, the monomeric glutamyl-tRNA synthetase from Bacillus subtilis can aminoacylate in vitro with glutamate both tRNAGlu and tRNAGln from B. subtilis and tRNAGln1 but not tRNAGln2 or tRNAGlu from Escherichia coli. The Km and Vmax values of the enzyme for its substrates in these homologous or heterologous aminoacylation reactions are very similar. This enzyme is the only aminoacyl-tRNA synthetase reported to aminoacylate with normal kinetic parameters two tRNA species coding for different amino acids and to misacylate at a high rate a heterologous tRNA under normal aminoacylation conditions. The exceptional lack of specificity of this enzyme for its tRNAGlu and tRNAGln substrates, together with structural and catalytic peculiarities shared with the E. coli glutamyl- and glutaminyl-tRNA synthetases, suggests the existence of a close evolutionary linkage between the aminoacyl-tRNA synthetases specific for glutamate and those specific for glutamine. A comparison of the primary structures of the three tRNAs efficiently charged by the B. subtilis glutamyl-tRNA synthetase with those of E. coli tRNAGlu and tRNAGln2 suggests that this enzyme interacts with the G64-C50 or G64-U50 in the T psi stem of its tRNA substrates.