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1.
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.  相似文献   

2.
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.  相似文献   

3.
Li KK  Beeson WT  Ghiviriga I  Richards NG 《Biochemistry》2007,46(16):4840-4849
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.  相似文献   

4.
Purification and characterization of beef pancreatic asparagine synthetase   总被引:1,自引:0,他引:1  
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.  相似文献   

5.
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.  相似文献   

6.
Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I   总被引:2,自引:0,他引:2  
The mechanism of Ca2+ inhibition of carbamylphosphate synthetase I has been investigated using purified enzyme obtained from livers of rats fed a high protein diet. Binding of Mn2+ to the enzyme was measured by EPR techniques at pH 7.8, and Scatchard plots of the data indicated one Mn2+-binding site with a K'd of 13 microM. From competition studies between Mn2+ and Ca2+ or Mg2+ binding, values of 180 microM were obtained for K'd (Mg) and 193 microM for K'd (Ca). A nonlinear least squares curve fitting program was used to calculate the K'm for MgATP2- at the metal-nucleotide binding sites using a simplified rate equation of the enzyme reaction mechanism. Values of 140 and 2420 microM were obtained for K'm (MgATP) at the first and second sites, respectively, at pH 7.8, with a free Mg2+ of 1 mM and other substrates and activators present at saturating concentrations. Variations of the bicarbonate, N-acetylglutamate, and ammonia concentrations in the absence and presence of different amounts of total calcium, from which free Ca2+, free Mg2+, MgATP2-, and CaATP2- concentrations were calculated, permitted values for K'i (CaATP) to be obtained by graphic procedures. Mean values of 375 and 120 microM were obtained for K'i (CaATP) at the first and second sites, respectively. Using the above kinetic constants, a computer model of the enzyme reaction was constructed and tested using two further sets of kinetic data obtained by varying the concentrations of Mg2+, Ca2+, MgATP2-, and CaATP2-. Poor fits were obtained unless the formation of a mixed complex involving CaATP2- competition with MgATP2- at the second metal-nucleotide-binding site was incorporated into the rate equation. Nonlinear least squares curve fitting of both sets of experimental data gave a well determined value of 124 microM for this final CaATP2- inhibitory constant. Sensitivity tests for variation of the primary kinetic constants with the computer model showed that the inhibitory effect of free Ca2+ was weak and that the observed calcium inhibition of carbamylphosphate synthetase can be accounted for primarily by competitive interaction of CaATP2- at the second MgATP2- binding site. With 1 mM free Mg2+ and 5 mM MgATP2-, half-maximal inhibition of enzyme activity was obtained with 0.2 mM CaATP2-.  相似文献   

7.
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.  相似文献   

8.
Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo-L-norleucine (DON) and 5-chloro-4-oxo-L-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.  相似文献   

9.
10.
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.  相似文献   

11.
The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.  相似文献   

12.
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.  相似文献   

13.
A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.  相似文献   

14.
MgATP substantially inhibited 1-alkyl-sn-glycero-3-phosphate (AGP) acetyltransferase found in neuronal nuclei. Other nucleotides and the ATP analogue AMP-PNP did not show a comparable inhibition. MgATP inhibition decreased in the presence of bovine serum albumin or the fatty acyl CoA synthetase inhibitor, Triacsin C. MgATP inhibition increased when nuclei were preincubated in 50 mM Tris-HCl (pH 7.4)/1 mM MgCl(2) at 37 degrees C, and preincubations elevated levels of nuclear free fatty acid. Exogenous free fatty acid, added to the acetylation incubations, increased the inhibition seen in the presence of MgATP. Oleoyl CoA, in the absence of MgATP, also inhibited AGP acetylation. These results suggested that MgATP supported the conversion of nuclear free fatty acids to fatty acyl CoA. Fatty acyl CoA may directly inhibit nuclear AGP acetyltransferase, but inhibition brought about by MgATP was competitive for the AGP substrate, suggesting an inhibitor close in structure to AGP. 1-Hexadecyl-2-arachidonoyl-sn-glycero-3-phosphate was identified as a competitive inhibitor for AGP in the acetylation reaction. Neuronal nuclei can convert AGP to 1-alkyl-2-acyl-sn-glycero-3-phosphate (AAcylGP), a reaction dependent upon MgATP and the presence of acetyl CoA or free CoA. This nuclear acylation was increased by free fatty acid addition and was seen using oleoyl CoA in the absence of MgATP. Nuclear AAcylGP formation was inhibited by bovine serum albumin and by Triacsin C. Thus, nuclear AGP acetyltransferase may be regulated by AGP acyltransferase activity and the availability of MgATP, a nucleotide that is rapidly lost during brain ischemia.  相似文献   

15.
Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.  相似文献   

16.
Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn''t affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.  相似文献   

17.
In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.  相似文献   

18.
Bachmann BO  Townsend CA 《Biochemistry》2000,39(37):11187-11193
Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.  相似文献   

19.
The kinetic mechanism of asparagine synthetase from rat liver has been studied. The mechanism of the reaction in the presence of high concentrations of total Mg2+ (50 mM) was suggested to be a uni-uni-bi-ter ping-pong-type without abortive complexes; glutamine binds first followed by glutamate release, and aspartate and ATP bind in order followed by ordered release of PPi, AMP, and asparagine. But, it is indicated that in the presence of 0.5-2.0 mM excess Mg2+ over ATP the binding of substrates after the release of glutamate is in a rapid equilibrium system such as ordered Mg2+ and random aspartate-MgATP. Mg2+ was demonstrated to have two roles in the catalysis; to modify the enzyme and to form a complex of MgATP.  相似文献   

20.
Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.  相似文献   

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