Catalytic cooperativity and subunit interactions in E. coli glutamine synthetase binding and kinetics with methionine sulfoximine and related inhibitors

Wedler, Frederick C.; Sugiyama, Yuichi; Fisher, Kathryn E.

doi:10.1021/bi00538a028

Cited by 21 publications

(15 citation statements)

References 29 publications

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“…Inactivation of E. coli GS by methionine sulfoximine induces a conformational change in the enzyme subunits and reduces the binding affinity for methionine sulfoximine in the remaining active subunits (11,13,20). This mechanism is consistent with our observation that addition of a second sample of T-P-L to partially inactivated GS did not restore the initial rate of inactivation (Fig.…”

Section: Self-protection Of P Syringae Subsp "Tabaci"supporting

confidence: 88%

“…"tabaci" Tox+ (Ptll3; University of Wisconsin), a virulent tabtoxin-producing strain, and P. syringae subsp. "tabaci" Tox-(Pa 45), a non-toxin-producing strain, were grown in shake culture at 25°C for 24 h in medium containing 30 mM K2HPO4, 7 mM KH2PO4, 10 mM NaCl, 20 [21]) and supplemented with an additional 20 mM (NH4)2SO4 (ammonia shock) during the stationary phase (1). Ammonia shock and harvesting were done during the stationary phase to maximize the adenylylation state.…”

Section: Methodsmentioning

confidence: 99%

“…GS (E10.5) was incubated with 10 IxM T-P-L, and GS activity was assayed as described in the legend to Fig. 1, except at t = 30 min the sample was divided into two samples, and additional T-P-L was added to the second sample to give 20 ,uM T-P-L. This time was defined as t0 and was used to replot the loss of GS activity in the two samples (curves 2 and 3).…”

Section: Methodsmentioning

confidence: 99%

“…4). Adenylylation also induces changes in the binding of glutamate (14) and methionine sulfoximine (20). Increasing the adenylylation state of subsp.…”

Section: Self-protection Of P Syringae Subsp "Tabaci"mentioning

confidence: 99%

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Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Knight¹,

Durbin²,

Langston-Unkefer³

1986

J Bacteriol

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Selected pathovars of Pseudomonas syringae produce an extracellular phytotoxin, tabtoxinine-p-lactam, that irreversibly inhibits its known physiological target, glutamine synthetase (GS). Pseudomonas syringae subsp. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection mechanism(s) used by these pathovars, we have determined that GS becomes adenylylated after toxin production is initiated and that the serine released from the zinc-activated hydrolysis of tabtoxin is a factor in the initiation of this adenylylation. The adenylylation state of this GS was estimated to range from E5. 7.*5. The irreversible inactivation by tabtoxinine-B-lactam of unadenylylated and adenylylated glutamine synthetase purified from P. syringae subsp. "tabaci" was investigated. Adenylylated GS was inactivated by tabtoxinine-p-lactam at a slower rate than was unadenylylated enzyme. Adenylylated GS (E7.510.5) was significantly protected from this inactivation in the presence of the enzyme effectors, AMP, Ala, Gly, His, and Ser. Thus, the combination of the adenylylation of GS after toxin production is initiated and the presence of the enzyme effectors in vivo could provide part of the self-protection mechanism used by subsp. "tabaci".

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Section: Self-protection Of P Syringae Subsp "Tabaci"supporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…4). Adenylylation also induces changes in the binding of glutamate (14) and methionine sulfoximine (20). Increasing the adenylylation state of subsp.…”

Section: Self-protection Of P Syringae Subsp "Tabaci"mentioning

confidence: 99%

See 2 more Smart Citations

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Knight¹,

Durbin²,

Langston-Unkefer³

1986

J Bacteriol

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“…[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] Much effort has been invested in developing GS inhibitors not only for antibacterial research, but also for weed control, 20,25,[30][31][32] since GS is a key enzyme in ammonia assimilation in plants. However, no inhibitor has shown improved activity compared to MSO or PPT.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

Nordqvist

Nilsson

Röttger

et al. 2008

Bioorganic & Medicinal Chemistry

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Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery. Bioorganic & Medicinal AbstractA combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC 50 of 610  15 µM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.

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Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy

1996

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Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H 2 0 and D 2 0 solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 2 0.5 and 4.6 ? 0.5 water molecules coordinated to Eu3+ bound to the nl site of unadenylylated enzyme, GSo, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 ? 0.5 water molecules coordinated to Eu3+ at site n2 for both GSo and GSl2. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 ? 1.5 A. Distances between a Tb" ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site nl was observed to decrease from Kd = 0.35 2 0.09 pM for GSo to Kd = 0.06 ? 0.02 pM for GSI2. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 2 0.9 pM and Kd = 2.6 2 0.7 pM for GSo and GSI2, respectively. Competition experiments indicate that Mn2+ affinity at site nl decreases as a function of increasing adenylylation state, from Kd = 0.05 2 0.02 pM for GSo to K d = 0.35 ? 0.09 pM for GSI2. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 ? 1.3 pM for GSo and Kd = 4.0 ? 1.0 pM for GSI2). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991. Biochemistry 30: 1413-141 8), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.

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Catalytic cooperativity and subunit interactions in E. coli glutamine synthetase binding and kinetics with methionine sulfoximine and related inhibitors

Cited by 21 publications

References 29 publications

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy

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