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

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α- -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-β-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 K_m values of the human lymphoblast enzyme are 0.46 m for glutamine and 0.71 m for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo- -norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.     

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Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.     

Mutational analysis of the glutamine phosphoribosylpyrophosphate amidotransferase pro-peptide

Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region. These mutations affect processing and enzyme activities. Processing of the leader is dependent upon glutamic acid residues at positions -2 and -1 as well as Cys1. In addition, processing requires a pro-peptide longer than 3 residues. Function of the active site cysteine is dependent on pro-peptide processing. Enzyme purified from a pro-peptide deletion strain has activity and iron content that is comparable to the wild type. These results establish that the pro-peptide is not essential for enzyme maturation, but they leave unanswered the question of pro-peptide function.     

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.     

Affinity chromatography of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Purified glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis bound to affinity adsorbents containing immobilized adenine nucleotides. Although the enzyme probably bound via an allosteric site at which AMP acts most effectively, 50 times more enzyme was bound by N⁶-(aminohexyl)-ATP-agarose than by N⁶-(aminohexyl)-AMP-agarose. The enzyme could be efficiently and specifically eluted from N⁶-(aminohexyl)-ATP-agarose with the substrate phosphoribosylpyrophosphate, which antagonizes AMP inhibition in kinetic experiments. Elution could also be effected by 0.5 m KCl or by chelation of Mg²⁺ ions. The usefulness of these techniques in purification of partially purified amidotransferase was demonstrated.     

Mutagenesis of ligands to the [4 Fe-4S] center of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Several mutations were constructed in residues thought to provide ligands for a [4Fe-4S] cluster in Bacillus subtilis amidophosphoribosyltransferase using site-directed mutagenesis of cloned purF. These replacements confirm the identification of cysteinyl ligands to the Fe-S center. Of five mutant enzymes, two had no activity, two less than 25% of the wild type activity, and one was lethal and could not be studied. The Fe content of the two mutant enzymes with partial activity was similar to that of the wild type. Results of partial characterization suggest that the [4Fe-4S] cluster is not involved in allosteric regulation and does not play a specific role in the ammonia- or glutamine-dependent reactions of the enzyme. At least partial enzymatic activity is required for NH2-terminal processing. Pulse labeling experiments suggest that processing is a slow post-translational process which is dependent upon cellular factors. A relationship between Fe-S centers and NH2-terminal processing of an undecapeptide leader suggests a functional connection between these two structural elements in amidophosphoribosyltransferase.     

Role of NifS in maturation of glutamine phosphoribosylpyrophosphate amidotransferase.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is synthesized as an inactive precursor that requires two maturation steps: incorporation of a [4Fe-4S] center and cleavage of an 11-residue NH2-terminal propeptide. Overproduction from a multicopy plasmid in Escherichia coli leads to the formation of soluble proenzyme and mature enzyme forms as well as a small fraction of insoluble proenzyme. Heterologous expression of Azotobacter vinelandii nifS from a compatible plasmid increased the maturation of the soluble proenzyme three- to fourfold without influencing the content of the insoluble fraction. These results support a role for NifS in heterologous Fe-S cluster assembly and enzyme maturation.     

Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo

Glutamine phosphoribosylpyrophosphate amidotransferase is stable in growing cells, but is inactivated in an oxygen-dependent process at various rates in starving or antibiotic-treated cells. On the basis of studies of the purified enzyme, we suggested (D.A. Bernlohr and R.L. Switzer, Biochemistry 20:5675-5681, 1981) that the inactivation in vivo was regulated by substrate stabilization and a competition between stabilizing (AMP) and destabilizing (GMP, GDP, and ADP) nucleotides. This proposal was tested by measuring the intracellular levels of these metabolites under cultural conditions in which the stability of the amidotransferase varied. The results established that the stability of amidotransferase in vivo cannot be explained by the simple interactions observed in vitro. Metabolite levels associated with stability of the enzyme in growing cells did not confer stability under other conditions, such as ammonia starvation or refeeding of glucose-starved cells. The data suggest that a previously unrecognized event, possibly a covalent modification of amidotransferase, is required to mark the enzyme for oxygen-dependent inactivation.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.     

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in vitro inactivation.

The oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase (ATase) is demonstrated in cell extracts of Bacillus subtilis. The rate of inactivation of ATase in vitro is apparently first order with respect to oxygen concentration and ATase activity. ATase inactivation in vitro (or in vivo) cannot be reactivated by a variety of reductants. ATase is significantly stabilized to oxygen-dependent inactivation in vitro in the presence of tetrasodium phosphoribosylpyrophosphate and glutamine together. The effects of the end product inhibitors, adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP), on the stability of ATase are antagonistic. AMP stabilizes ATase, whereas GMP destabilizes the enzyme. The stability of ATase can be manipulated over wide ranges by variations in the AMP/GM ratio. The effects of AMP and GMP on the inactivation of ATase in vitro are very specific. ATase is partially inhibited by 1,10-phenanthroline, suggesting that the enzyme contains iron (or some other chelatable metal ion). The inactivation of ATase in vitro is proposed to present a model for the reconstruction of the inactivation of ATase in stationary-phase cells of B. subtilis.     

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

Glutamine amidotransferase function. Replacement of the active-site cysteine in glutamine phosphoribosylpyrophosphate amidotransferase by site-directed mutagenesis

Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.     

Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Native Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase contains a [4Fe-4S] cluster in the diamagnetic (+2) state. The cluster is essential for catalytic function, even though amidotransferase does not catalyze a redox reaction. The ability of the Fe-S cluster to undergo oxidation and reduction reactions and the consequences of changes in the redox state of the cluster for enzyme activity were studied. Treatment of the enzyme with oxidants resulted in either no reaction or complete dissolution of the Fe-S cluster and loss of activity. A stable +3 oxidation state was not detected. A small amount of paramagnetic species, probably an oxidized 3Fe cluster, was formed transiently during oxidation. The native cluster was poorly reduced by dithionite, but it could be readily reduced to the +1 state by photoreduction with 5-deazaflavin and oxalate. The reduced enzyme did not display an EPR spectrum typical of [4Fe-4S] ferredoxins in the +1 state, unless it was prepared under denaturing conditions. M?ssbauer spectroscopy of reduced 57Fe-enriched amidotransferase confirmed that the cluster was in the +1 state, but the magnetic properties of the reduced cluster observed at 4.2 K indicated that it is characterized by a ground state spin S greater than or equal to 3/2. The midpoint potential of the +1/+2 couple was too low to measure accurately by conventional techniques, but it was below -600 mV, which is 100 mV more negative than reported for [4Fe-4S] clusters in bacterial ferredoxins. Fully reduced amidotransferase had about 40% of the activity of the native enzyme in glutamine-dependent phosphoribosylamine formation. The fact that both the +1 and +2 forms of the enzyme are active indicates that the cluster does not function as a site of reversible electron transfer during catalysis.