Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

Rodriguez, Evelyn J.; Lander, Noelia; Ramı́rez, José Luis

doi:10.1590/s0074-02762009000500014

Cited by 9 publications

(10 citation statements)

References 8 publications

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“…The intersecting lines of Lineweaver-Burk's double reciprocal plots (Figures 1 and 2) clearly indicated that the affinity of the enzyme for one substrate was independent of the concentration of the cosubstrate. Similar results were reported for the enzyme of T. cruzi [20] However, for the enzyme in M. expansa and T. brucei [36], the K m value of Mg-ATP 2- has been shown to increase at higher F-6-P concentration. The K cat value (320 ± 15 sec −1 ) for F-6-P as recorded for S. cervi PFK in present investigation was found to be similar to that from E. histolytica (344 sec −1 ) [37] and about five times higher than that of T. brucei (61 sec −1 ) [38].…”

Section: Discussionsupporting

confidence: 85%

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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Section: Discussionsupporting

confidence: 85%

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

Sharma

2011

Enzyme Research

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“…Several glycosomal enzymes found in our proteome analyses, like hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) (Shih et al , 1998) and pyruvate phosphate dikinase (PPDK) (Bringaud et al , 1998, Maldonado & Fairlamb, 2001, Shih et al , 1998) produce or utilize PPi. Phosphofructokinase (PFK) is an ATP-dependent enzyme but shows a high degree of sequence and structural similarity with PPi-dependent enzymes (Rodriguez E., 2009, Michels et al , 1997, McNae et al , 2009). Thus, interaction with potential PPi- or polyP-binding sites could explain their pull down by polyP.…”

Section: Discussionmentioning

confidence: 99%

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids

Negreiros

Lander

Huang

et al. 2018

Molecular Microbiology

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Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

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“…The T. cruzi PFK presented Michaelian kinetics for fructose 6-phosphate and was activated only by 5′-AMP [19]. In addition, like the similar enzyme from other trypanosomatids, T. cruzi PFK shows the highest sequence similarity with PP i -dependent PFK, despite being ATP-dependent, and is glycosomal [20].…”

Section: Glycolytic Pathwaymentioning

confidence: 90%

Glucose metabolism in Trypanosoma cruzi

Maugeri

Cannata

Cazzulo

2011

Essays in Biochemistry

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The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major pathways: glycolysis and the pentose phosphate pathway. Glucose is taken up via one facilitated transporter and its catabolism by the glycolytic pathway leads to the excretion of reduced products, succinate and l-alanine, even in the presence of oxygen; the first six enzymes are located in a peroxisome-like organelle, the glycosome, and the lack of regulatory controls in hexokinase and phosphofructokinase results in the lack of the Pasteur effect. All of the enzymes of the pentose phosphate pathway are present in the four major stages of the parasite's life cycle, and some of them are possible targets for chemotherapy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase are present, but there is no reserve polysaccharide.

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Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

Cited by 9 publications

References 8 publications

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

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

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids

Glucose metabolism in Trypanosoma cruzi

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