Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-alpha-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction. 5-beta-phosphoribosyl-1-amine, using [1-14C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent Km values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.     

Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties.

Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.     

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.     

Purification and properties of the arginine-specific carbamoyl-phosphate synthase from Saccharomyces cerevisiae.

The arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (Km = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.     

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.     

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 properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

Independent localization and regulation of carbamyl phosphate synthetase A polypeptides of Neurospora crassa

Summary Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon argininestarvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.     

Enhancement of the glutaminase activity of carbamyl phosphate synthetase by alterations in the interaction between the heavy and light subunits.

Glutamine-dependent carbamyl phosphate synthetase (from Escherichia coli) was previously shown to be composed of a light subunit (molecular weight similar to 42,000) which has the binding site for glutamine and a heavy subunit (molecular weight similar to 130,000) which has binding sites for the other reactants and allosteric effectors. The subunits may be separated with retention of catalytic activities; only the separated light subunit exhibits glutaminase activity. The previous finding that storage of the native enzyme at pH 9 at 0 degrees increased its glutaminase activity by about 25-fold was further investigated; such storage markedly decreased the glutamine- and ammonia-dependent synthetase activities of the enzyme. Treatment of the enzyme with p-hydroxymercuribenzoate led to transient increase of glutaminase activity followed by inhibition. When the enzyme was treated with N-ethylmaleimide or with 5,5'-dithiobis-(2-nitrobenzoate), the glutaminase activity was increased by about 250-fold with concomitant loss of synthetase activities. The enhancement of glutaminase produced by storage of the enzyme at pH 9 was associated with intermolecular disulfide bond formation and aggregation of the enzyme. Aggregation also was observed after extensive treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) or N-ethylmaleimide. However, a moderate increase of glutaminase activity (about 30-fold) was observed without aggregation under conditions in which one sulfhydryl group on the light subunit reacted with either reagent. The findings suggest that the increased glutaminase activities observed here are associated with structural changes in the enzyme in which the intersubunit relationship is altered so as to uncouple the catalytic functions of the enzyme and to facilitate access of water to the glutamine binding site on the light subunit.     

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.     

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Ornithine transcarbamylase of rat liver. Kinetic, physical, and chemical properties.

Ornithine transcarbamylase of rat liver has been purified to homogeneity. The purified enzyme of specific activity 870 to 920 focuses as a single protein at pH 7.2. At pH 7.7, the Km for carbamyl phosphate is 0.026 mM, and the Km for ornithine is 0.04 mM. The inhibition constants of a number of amino acids that act as competitive inhibitors of the enzyme are reported. The native enzyme of Mr = 112,000 is composed of three subunits of Mr = 39,600 +/- 1,000. Chemical evidence indicates that the subunits are identical in amino acid composition and amino acid sequence. The amino acid sequence of the NH2-terminal region of ornithine transcarbamylase is Ser-Gln-Val-Gln-Leu-Lys-Gly-Ser-Asp-Leu-Leu-Thr-Leu-Lys-Asn-(Phe)-X-Thr-X-Glu-Ile-Gln-Tyr-Met-.     

Structure-Function Relationships in the Arginine Pathway Carbamoylphosphate Synthase of Saccharomyces cerevisiae

The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was shown to be highly unstable and could not be substantially purified. In spite of this instability, a number of important properties of this enzyme were determined with crude preparations. A molecular weight of 140,000 (7.9S) was estimated for the native enzyme by sucrose gradient centrifugation; a significantly higher value, 175,000, was obtained by gel filtration on Sephadex. The enzyme is an aggregate consisting of two protein components, coded for by the unlinked genes cpaI and cpaII. These components were separated by diethylaminoethyl-cellulose chromatography. Their molecular weights, estimated by Sephadex gel filtration, were 36,000 and 130,000. The large component catalyzed the synthesis of carbamoylphosphate from ammonia. The small component was required in addition to the large one for the physiologically functional glutamine-dependent activity. Apparent Michaelis constants at pH 7.5 of 1.25 mM for glutamine and 75 mM for NH(4)Cl were measured with the native enzyme. The use of various glutamine analogs, including 2-amino-4-oxo-5-chloropentanoic acid, indicated that binding of glutamine to a site located on the small component was followed by transfer of its amide nitrogen to the ammonia site on the heavy component. This ammonia site was able to function independently of the utilization of glutamine. However, binding of glutamine was conjectured to cause a conformational change in the heavy component that greatly increased the rate of synthesis of carbamoylphosphate from ammonia. Glutamine, which was also shown to stabilize the aggregation of the two components, appeared to be a major effector of the catalytic and structural properties of CPSase A. In view of these observations, the CPSase A of yeast appears to share a number of structural and catalytic properties with the Escherichia coli enzyme. Obviously, the unlinked cpaI and cpaII genes of yeast are homologous to the adjacent carA and carB genes that code for the two subunits of the bacterial enzyme.     

The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli.

Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.     

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.     

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.     

Studies on glutamine synthetase. Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) fromPhaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. TheK _m values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0.5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.