Activation of Thermus phosphofructokinase by monovalent cations

Stellwagen, Earle; Thompson, Sioe Thung

doi:10.1016/0005-2744(79)90075-5

Cited by 6 publications

(3 citation statements)

References 7 publications

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“…The electron-dense ion T1+ (80 electrons) has been employed to study effects of monovalent cations on protein activity (Suelter & Snell, 1977;Stellwagen & Thompson, 1979;Hill & Castellino, 1986;Takada et al, 1990) and on ion channels (Urban et al, 1980;Zeiske & van Driessche, 1983;van Driessche & Zeiske, 1985), and to probe the binding sites of monovalent cations (Urry et al, 1982;Markham, 1986;Hill et al, 1987;Gursky et al, 1992). In all these studies of effects of monovalent cations on enzymes, ion carriers, and ion channels, T1+ has been found to bind at known K + or NH4+ binding sites.…”

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confidence: 99%

Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

1995

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Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, T1+ and cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. unit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that T1+, Cs+, and NH4+ are competitive inhibitors to N H 2 0 H in the y-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites ( n l , n 2 ) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n l and n2 is 6 A, between n, and TI+ is 4 A, and between n2 and TI+ is 7 A . Glu 212 and the substrate glutamate are bridging ligands for the n , ion and Tl'. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the TI' binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form y-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the y-glutamyl phosphate to yield Gln. ( 5 ) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate y-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.Keywords: glutamine synthetase; NH,+; T1+ Glutamine synthetase (GS) is a primary biological catalyst in the sense that it catalyzes the first step at which nitrogen is brought into cellular metabolism: glutamate + NH4' + ATP + glutamine + ADP + Pi. The product glutamine is a source of nitrogen in the biosynthesis of many other me...

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confidence: 99%

Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

1995

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“…Another possibility is the changes in metabolic enzymes caused by K + . Phosphofructokinases of both prokaryotes and eukaryotes are activated in the presence of K + 3435. Generally, phosphofructokinase participates in a rate-limiting step of glycolysis, but pyruvate kinase activity is also important for determining the flux of sugar catabolism in eukaryotes36; pyruvate kinase is known to be activated by K + 37.…”

Section: Discussionmentioning

confidence: 99%

Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria

Ueda

Kawamura

Iijima

et al. 2016

Sci Rep

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Potassium (K+) is an essential macronutrient for all living organisms including cyanobacteria. Cyanobacteria are a group of bacteria performing oxygenic photosynthesis, widely studied in basic and applied sciences. The primary metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is altered by environmental conditions, and it excretes organic acids and hydrogen under dark, anaerobic conditions. Here we demonstrated that K+ widely changes the primary carbon metabolism of this cyanobacterium. Succinate and lactate excretion from the cells incubated under dark, anaerobic conditions was enhanced in the presence of K+, while hydrogen production was repressed. The addition of K+ and the genetic manipulation of acetate kinase AckA and an RNA polymerase sigma factor SigE additively increased succinate and lactate production to 141.0 and 217.6 mg/L, which are 11 and 46 times, compared to the wild-type strain without K+, respectively. Intracellular levels of 2-oxoglutarate, succinate, fumarate, and malate increased by K+ under dark, anaerobic conditions. This study provides the evidence of the considerable effect of K+ on the biosynthesis of anionic metabolites in a unicellular cyanobacterium.

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“…(5,21). Na+ (added as NaCI) was ineffective as a stimulator of the enzymes from rabbit muscle (20) and from the thermophile Thermus (22).…”

Section: Mm)mentioning

confidence: 99%

Effect of Salts on the Activity of Carrot Phosphofructokinase

Turner¹,

Tomlinson²,

Caldwell³

1980

Plant Physiol.

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Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts (10,11,14,15) and this inhibition is cooperative (16). A characteristic property of phosphofructokinase from higher plants is the strong cooperative inhibition given by very low concentrations of P-enolpyruvate (16). With the pea seed enzyme, 50% inhibition of phosphofructokinase activity may be obtained with P-enolpyruvate concentrations as low as 1.3 uM (16). It is believed that Penolpyruvate is a major factor in the regulation of plant phosphofructokinase and hence in the control of glycolysis (23,24). Pea seed phosphofructokinase is also strongly inhibited by low concentrations of 2-P-glycerate and 3-P-glycerate (17).In an earlier communication it was shown that NaCl and KCI stimulated the activity of phosphofructokinase from carrot roots (7). The carrot enzyme was strongly inhibited by P- Partial Purification of Carrot Root Phosphofructokinase. The method of preparation of carrot phosphofructokinase was a modification of that described previously (7). Approximately I kg fresh carrots were peeled and placed in a juice extractor. The juice was treated with 0.33 volume of a mixture of 0.1 M Tris, 50 mM NaF, 10 mM MgCI2 and 8 mm EDTA which had been adjusted to pH 8.0 with acetic acid (buffer A), and centrifuged at 20,000g for 10 min. After filtering through glass wool, the supernatant was adjusted to pH 8.0 with NH40H and saturated (NH4)2SO4 (pH 8.0) added to 35% saturation. The precipitate was removed by centrifugation at 20,000g for 20 min and the supernatant raised to 55% saturation with (NH4)2SO4. The mixture was centrifuged at 20,000g for 20 min and the pellet resuspended in a I in 10 dilution of buffer A (buffer B) containing I mm ATP and heated at 55 C for 3 min. After cooling and centrifugation at 27,000g for 15 min the supernatant was again fractionated with (NH4)2SO4. The pellet obtained between 25% and 35% saturation was resuspended in 6 ml of buffer B containing 0.1 mm ATP and 0.1 mm fructose-1,6-P2 and dialyzed against buffer B. The dialyzed material was frozen and thawed twice and the resultant precipitate removed by centrifugation. This preparation had a specific activity of approximately 0.7 Amol fructose-1,6-P2 produced/min mg protein which represented a 20-fold purification from the crude extract. Phosphatases acting on fructose-6-P, fructose-1,6-P2 and ATP were not detected in this partially purified preparation.Assay of Phosphofructokinase Activity. Enzyme activity was assayed by coupling the production of fructose-1,6-P2 with the oxidation of NADH through aldolase, triose-P isomerase and aglycero-P dehydrogenase. (NH4)2SO4 was removed from the coupling enzymes before use by dialysis against buffer B. Standard assay reaction mixtures contained in a total volume of 3 ml, 0.75 ,Lmol ATP, 4.8 t,mol MgCI2, 0.42 ,tmol 8-NADH, 0.3 unit aldolase, 15 units triose-P isomerase, 3 units a-glycero-P d...

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Activation of Thermus phosphofructokinase by monovalent cations

Cited by 6 publications

References 7 publications

Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria

Effect of Salts on the Activity of Carrot Phosphofructokinase

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