Interdomain Signaling in Glutamine Phosphoribosylpyrophosphate Amidotransferase

Bera, Amit; Chen, Sihong; Smith, Janet L.; Zalkin, Howard

doi:10.1074/jbc.274.51.36498

Cited by 22 publications

(17 citation statements)

References 11 publications

(23 reference statements)

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“…Hence, in addition to suggesting that βAspAMP formation commits the enzyme to asparagine synthesis, implying that ammonia transfer from the N-terminal domain through the intramolecular tunnel is totally efficient, the model provides evidence for the hypothesis that glutamine can bind to the E.Asp.ATP ternary complex to yield a quaternary complex from which glutamate and ammonia can be released. Thus, coordination of catalytic activity in the two active sites of AS-B during glutamine-dependent asparagine synthesis appears to be remarkably small prior to βAspAMP formation, which is in sharp contrast to the coupling of glutaminase and synthetase activities seen for other Ntn glutamine-dependent amidotransferases (73)(74)(75). The lack of ATP/PP i exchange has been rationalized by assuming that PP i is released as the final product from the enzyme.…”

Section: Kinetic Mechanism Of Asparagine Synthetasementioning

confidence: 96%

Asparagine Synthetase Chemotherapy

Richards

Kilberg

2006

Annu. Rev. Biochem.

201

221

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Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzymebased methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

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Section: Kinetic Mechanism Of Asparagine Synthetasementioning

confidence: 96%

Asparagine Synthetase Chemotherapy

Richards

Kilberg

2006

Annu. Rev. Biochem.

201

221

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“…This signaling interaction repositions the glutamine loop and moves Arg-73 for an optimal salt bridge with the carboxyl of glutamine, which is required for high affinity glutamine binding. The interdomain signaling steps that activate the glutamine site in response to PRPP binding have recently been monitored through changes in intrinsic tryptophan fluorescence in enzymes engineered to contain single tryptophan reporters (6,7). Interestingly, replacements of Ile-335 or Tyr-74 were shown not only to perturb the interdomain signaling that activates the glutamine site but also to perturb channel function.…”

mentioning

confidence: 99%

Dual Role for the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel

Bera¹,

Smith²,

Zalkin³

2000

Journal of Biological Chemistry

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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-Å 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. Glutamine PRPP1 amidotransferase catalyzes the first reaction in the pathway for de novo purine nucleotide synthesis and is the key regulatory enzyme in the pathway. The enzyme is a member of an Ntn, N-terminal nucleophile family of glutamine amidotransferases (1). The overall reaction, shown by Equation 1, takes place in two steps at active sites in two domains.An N-terminal glutaminase domain is responsible for hydrolysis of glutamine (Equation 2). NH 3 derived from glutamine hydrolysis reacts with PRPP at a site in the C-terminal PRTase domain (Equation 3). Crystal structures have been determined for a ligand-free (2) and an enzyme-substrate analog ternary complex (3). In the ligand-free enzyme, denoted state I, the two active sites are separated by 16 Å, and neither of the sites is properly organized for catalysis. The PRPP site is open to the solvent such that bound substrate would be susceptible to hydrolysis. The glutamine site in the ligand-free enzyme is in a closed conformation, unfavorable for entry of glutamine, and Arg-73, a residue required for glutamine binding, is extensively hydrogen-bonded and unavailable for interaction with the glutamine ␣-carboxyl group. In the crystal structure of the ternary complex, designated state III, conformational changes have optimized the two sites for catalysis, and a 20-Å hydrophobic tunnel has formed to channel NH 3 derived from glutamine hydrolysis to the PRPP site. There are thus several functional consequences resulting from PRPP binding. First, binding of PRPP activates the glutamine site by lowering the K m for glutamine by 100-fold and increasing k cat by 3-fold (4). This interdomain signaling prevents the wasteful hydrolysis of glutamine in the absence of PRA synthesis. Formation of the NH 3 channel is a second consequence of PRPP bind...

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“…Inherent flexibility of the glutaminase active site, perhaps in concert with motion of the flexible loop, is thought to be responsible for basal glutaminase activity. In contrast, the ligand-free A. aeolicus enzyme is inferred to be folded in a stable, inactive conformation that requires PRPP binding to equilibrate with a conformation competent for glutaminase activity, a step referred to as interdomain signaling (1,4), and to form the NH 3 channel. The two steps, interdomain signaling and channel formation, are not as tightly linked as in the E. coli enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…The GPAT half reactions are coupled, and NH 3 is channeled by transient forms of the enzyme, in which closing of a flexible loop upon PRPP binding activates the glutaminase domain and forms an NH 3 channel between the active sites (1,2,4,11). PRA, the product of the second GPAT half reaction, is susceptible to hydrolysis to ribose 5-phosphate, with a half-life of 5 s under physiological conditions at 37°C (17).…”

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confidence: 99%

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

Bera¹,

Chen²,

Smith

et al. 2000

J Bacteriol

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Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH 3 from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH 3 for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.The first two steps in de novo purine nucleotide synthesis involve reactions in which labile enzyme intermediates are transferred between distal, noncontiguous sites. In the first reaction, catalyzed by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (GPAT), NH 3 , generated by hydrolysis of glutamine at an N-terminal glutaminase domain, is sequestered from H 2 O by transfer through a 20-Å tunnel to a C-terminal PRPP site where synthesis of phosphoribosylamine (PRA) takes place (11). Channeling of NH 3 through a hydrophobic tunnel prevents its release from the enzyme and also avoids the likelihood of protonation of NH 3 to NH 4 ϩ , which is not a substrate (13). These steps are shown by equations 1 and 2, and the synthesis of glycinamide ribonucleotide (GAR) by the second enzyme, GAR synthetase (GARS), is given in equation 3.Glutamine ϩ H 2 O 3 glutamate ϩ NH 3(1)The GPAT half reactions are coupled, and NH 3 is channeled by transient forms of the enzyme, in which closing of a flexible loop upon PRPP binding activates the glutaminase domain and forms an NH 3 channel between the active sites (1, 2, 4, 11). PRA, the product of the second GPAT half reaction, is susceptible to hydrolysis to ribose 5-phosphate, with a half-life of 5 s under physiological conditions at 37°C (17). The kinetics for the synthesis of GAR by Escherichia coli GPAT and GARS were found to be inconsistent with free diffusion of PRA between enzymes and, as a result, a direct-transfer mechanism was proposed (17). Since evidence for a GPAT-GARS physical interaction was not obtained, direct PRA transfer was assumed to involve a transient channeling interaction between GPAT and GARS. The transient...

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Interdomain Signaling in Glutamine Phosphoribosylpyrophosphate Amidotransferase

Cited by 22 publications

References 11 publications

Asparagine Synthetase Chemotherapy

Asparagine Synthetase Chemotherapy

Dual Role for the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

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