The roles of magnesium ions in the reaction catalysed by phosphofructokinase from <i>Trypanosoma brucei</i>

Cronin, Ciarán N.; Tipton, Keith F.

doi:10.1042/bj2470041

Cited by 17 publications

(10 citation statements)

References 36 publications

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“…S8). Similar inhibition has been observed for other ATP-dependent enzymes (19,20). Although the ATP concentration dependence could be readily fit by the requirement for two Mg 2ϩ ions at both 1 mM and 0.2 mM MgCl 2 (Fig.…”

supporting

confidence: 80%

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Taylor

Conrad

Wahl

et al. 2011

Journal of Biological Chemistry

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DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg 2؉ ions for catalysis and that an essential Mg 2؉ ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg 2؉ changes along the reaction coordinate. At saturating concentrations of ATP and Mg 2؉ ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg 2؉ , the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.Breaks in DNA result from spontaneous hydrolysis of the phosphodiester backbone and are formed as transient intermediates during DNA replication and repair pathways. Mammals have three genes encoding DNA ligases that seal these breaks and restore the continuous nature of chromosomes. DNA ligase I (LIG1) 2 is essential for ligation of single-strand breaks in the nucleus, including the ligation of Okazaki fragments during discontinuous DNA replication (1). DNA ligase III (LIG3) is required for mitochondrial DNA replication and repair. Although LIG3 has been assumed to play essential roles in nuclear DNA repair, it was recently shown to be dispensable for nuclear genomic maintenance (2, 3). DNA ligase IV (LIG4) is specialized for repair of nuclear double-strand breaks and is required for nonhomologous end joining and V(D)J recombination (4, 5).The overall reaction catalyzed by DNA ligases involves the formation of a phosphodiester bond between an adjacent 3Ј-hydroxyl and a 5Ј-phosphate in DNA. The reaction proceeds via a universally conserved pathway ( Fig. 1) (6 -8). First, the apoenzyme catalyzes transfer of the AMP group from a nucleotide cofactor to an active site lysine, forming an adenylylated enzyme intermediate. Eukaryotic ligases use ATP as the adenylyl group donor, whereas bacterial ligases utilize either ATP or NAD ϩ . After binding a nicked DNA substrate, the adenylylated enzyme catalyzes transfer of the adenylyl group to the 5Ј-phosphate present at the nick, forming an adenylylated DNA intermediate. In the fina...

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supporting

confidence: 80%

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Taylor

Conrad

Wahl

et al. 2011

Journal of Biological Chemistry

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“…Similar wide differences in K m values of Mg-ATP and F-6-P were also observed with the enzyme isolated from other parasites such as M. expansa [32], A. suum [15] T. brucei [21], and T. cruzi [20] whereas the enzyme from vertebrate tissues exhibited very less difference between the K m values of Mg-ATP and F-6-P [33]. The binding of the substrate Mg-ATP to PFKs from different origins shows a hyperbolic dependence with half saturation values in the range of 10–100 μ M in most of the cases.…”

Section: Discussionsupporting

confidence: 74%

Kinetic Characterisation of Phosphofructokinase Purified fromSetaria cervi: A Bovine Filarial Parasite

Sharma

2011

Enzyme Research

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Phosphofructokinase (PFK), a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P) or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that K m values for F-6-P (1.05 mM) and ATP (3 μM) were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM), exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n) value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP) competitively inhibited the filarial enzyme: K i and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP) also inhibited the enzyme competitively with the K i value equal to 0.8 mM. The Hill coefficient values (>1.5) for F-6-P (at inhibitory concentration of ATP) and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

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“…Only the enzymes catalyzing the last part of glycolysis, phosphoglycerate mutase, enolase and PYK are present in the cytosol. Glycolysis through the organelle seems unregulated: the activities of hexokinase and phosphofructokinase are not subject to regulation by the various effectors that are operational in many other cells [2,3]. In contrast, the activity of cytosolic PYK can be tightly controlled.…”

Section: Introductionmentioning

confidence: 99%

The putative effector‐binding site of Leishmania mexicana pyruvate kinase studied by site‐directed mutagenesis

et al. 2002

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The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P 2 ), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P 2 ). Based on the comparison of the threedimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P 2 present at the effector site (Rstate) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P 2 appeared to be entirely abrogated in the mutated enzymes confirming our predictions. ß

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The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei

Cited by 17 publications

References 36 publications

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Kinetic Characterisation of Phosphofructokinase Purified fromSetaria cervi: A Bovine Filarial Parasite

The putative effector‐binding site of Leishmania mexicana pyruvate kinase studied by site‐directed mutagenesis

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