Entamoeba histolytica: kinetic and molecular evidence of a previously unidentified pyruvate kinase

Saavedra, Emma; Olivos, Alfonso; Encalada, Rusely; Moreno‐Sánchez, Rafael

doi:10.1016/j.exppara.2004.01.009

Cited by 19 publications

(19 citation statements)

References 45 publications

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“…The reactions were always started with specific substrates, and basal activities in their absence were always subtracted. Amoebal PYK activity was measured in the presence of 0.2 m m F(1,6)P 2 as previously described [18]. In the presence of this activator, amoebal PYK displays the same activities at pH 6.0 and 7.0.…”

Section: Methodsmentioning

confidence: 99%

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

et al. 2007

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Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphatedependent (over ATP-dependent) enzymes. To determine the flux-control distribution of glycolysis and understand its underlying control mechanisms, a kinetic model of the pathway was constructed by using the software gepasi. The model was based on the kinetic parameters determined in the purified recombinant enzymes, and the enzyme activities, and steady-state fluxes and metabolite concentrations determined in amoebal trophozoites. The model predicted, with a high degree of accuracy, the flux and metabolite concentrations found in trophozoites, but only when the pyrophosphate concentration was held constant; at variable pyrophosphate, the model was not able to completely account for the ATP production ⁄ consumption balance, indicating the importance of the pyrophosphate homeostasis for amoebal glycolysis. Control analysis by the model revealed that hexokinase exerted the highest flux control (73%), as a result of its low cellular activity and strong AMP inhibition. 3-Phosphoglycerate mutase also exhibited significant flux control (65%) whereas the other pathway enzymes showed little or no control. The control of the ATP concentration was also mainly exerted by ATP consuming processes and 3-phosphoglycerate mutase and hexokinase (in the producing block). The model also indicated that, in order to diminish the amoebal glycolytic flux by 50%, it was required to decrease hexokinase or 3-phosphoglycerate mutase by 24% and 55%, respectively, or by 18% for both enzymes. By contrast, to attain the same reduction in flux by inhibiting the pyrophosphate-dependent enzymes pyrophosphate-phosphofructokinase and pyruvate phosphate dikinase, they should be decreased > 70%. On the basis of metabolic control analysis, steps whose inhibition would have stronger negative effects on the energy metabolism of this parasite were identified, thus becoming alternative targets for drug design.Abbreviations ADH, alcohol dehydrogenase; AK, adenylate kinase; ALDO, fructose 1,6-bisphosphate aldolase; AldDH, aldehyde dehydrogenase; ATP-PFK, ATP-dependent phosphofructokinase; DHAP, dihydroxyacetone phosphate; ENO, enolase; EtOH, ethanol; F6P, fructose 6-phosphate; F(1,6)P 2 , fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Gly3PDH, glycerol 3-phosphate dehydrogenase; HK, hexokinase; HPI, hexose 6-phosphate isomerase; HXT, hexose transporter; LDH, lactate dehydrogenase; MCA, metabolic control analysis; PGAM, 3-phosphoglycerate mutase; PGK, phosphoglycerate kinase; PGM, phosphoglucomutase; 3PGDH, 3-phosphoglycerate dehydrogenase; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PPi, pyrophosphate; PPi-PFK, pyrophosphate-dependent phosphofructokinase; PPP, pentose phosphate pathway; PFOR, pyruvate:ferredoxin oxidoreductase; PFOR-AldDH, lumped reacti...

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Section: Methodsmentioning

confidence: 99%

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

et al. 2007

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“…Although VOL. 20, 2007 THERAPEUTICS AGAINST AMITOCHONDRIATE PROTOZOA 167 it was reported earlier that E. histolytica lacks PK activity (255) and that pyruvate is synthesized either by PPDK or through the third pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase, and malic enzyme, a recent report demonstrated the presence of PK activity and a putative PK gene in E. histolytica (269). No PPDK activity has been detected in T. vaginalis (209), and pyruvate is synthesized predominantly by PK or through the oxaloacetate/malate pathway.…”

Section: Diversity In the Conversion Of Phosphoenolpyruvate To Acetylmentioning

confidence: 99%

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

2007

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SUMMARY The “amitochondriate” protozoan parasites of humans Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis share many biochemical features, e.g., energy and amino acid metabolism, a spectrum of drugs for their treatment, and the occurrence of drug resistance. These parasites possess metabolic pathways that are divergent from those of their mammalian hosts and are often considered to be good targets for drug development. Sulfur-containing-amino-acid metabolism represents one such divergent metabolic pathway, namely, the cysteine biosynthetic pathway and methionine γ-lyase-mediated catabolism of sulfur-containing amino acids, which are present in T. vaginalis and E. histolytica but absent in G. intestinalis. These pathways are potentially exploitable for development of drugs against amoebiasis and trichomoniasis. For instance, l-trifluoromethionine, which is catalyzed by methionine γ-lyase and produces a toxic product, is effective against T. vaginalis and E. histolytica parasites in vitro and in vivo and may represent a good lead compound. In this review, we summarize the biology of these microaerophilic parasites, their clinical manifestation and epidemiology of disease, chemotherapeutics, the modes of action of representative drugs, and problems related to these drugs, including drug resistance. We further discuss our approach to exploit unique sulfur-containing-amino-acid metabolism, focusing on development of drugs against E. histolytica.

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“…The higher incorporation of 32 P i into the enzyme in the presence of 32 PP i ϩ PEP ϩ AMP, compared with 32 PP i ϩ PEP (Table III), suggests that the phosphorylated enzyme produced in the first partial reaction (PEP 3 pyruvate) does not catalyze the second phosphorylation from 32 PP i in the absence of AMP. This finding suggested that the ternary complex EP-AMP-PP i was essential for the pyrophosphorylation of rEhPPDK.…”

Section: Resultsmentioning

confidence: 97%

“…Pyrophosphorylation of rEhPPDK with 32 PP i -It was found that 21 Ϯ 3% of rEhPPDK bound 32 P i covalently in the presence of 32 PPi ϩ PEP ϩ AMP in the reaction mixture and 5.7 Ϯ 0.7% with either 32 PP i , 32 PPi ϩ AMP or 32 PP i ϩ PEP (Table III). The higher incorporation of 32 P i into the enzyme in the presence of 32 PP i ϩ PEP ϩ AMP, compared with 32 PP i ϩ PEP (Table III), suggests that the phosphorylated enzyme produced in the first partial reaction (PEP 3 pyruvate) does not catalyze the second phosphorylation from 32 PP i in the absence of AMP.…”

Section: Resultsmentioning

confidence: 99%

“…Pyrophosphorylation of rEhPPDK with 32 PP i -The reaction was started by the addition of rEhPPDK (0.4 mg of protein, equivalent to 1.04 nmol) to 500 l of medium containing 50 mM imidazole (pH 6.3), 10 mM MgCl 2 , 4.5 mM NH 4 Cl, 2 mM 32 PP i (ϳ14 Ci; Perkin Elmer), and the indicated substrates. The reaction was stopped after 0.5-1.5 s of incubation at room temperature by the addition of 10% trichloroacetic acid.…”

Section: Kinetic Mechanismmentioning

confidence: 99%

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Kinetic Mechanism and Metabolic Role of Pyruvate Phosphate Dikinase from Entamoeba histolytica

Varela-Gómez

Moreno‐Sánchez

Pardo

et al. 2004

Journal of Biological Chemistry

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The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/ pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [ 32 P i ]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The ⌬G value for the reaction catalyzed by pyruvate phosphate dikinase (؉2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica. Pyruvate phosphate dikinase (PPDK)1 catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PP i ) to ATP, pyruvate (Pyr) and inorganic phosphate (P i ). The enzyme is found in bacteria such as Propionibacterium shermanii (1) and Clostridium symbiosum (2), in the mesophyll cells of C 4 plants (3), in leaves and roots of C 3 plants (4) and C 3 -C 4 intermediate plants (5), in the protists Entamoeba histolytica (6), Giardia lamblia (7), trypanosomatids, and Euglena (8), and in a thermophilic actinomyces microorganism (9). The functional role of PPDK depends on the organism. For example, it is well established that PPDK is responsible for the synthesis of the primary CO 2 acceptor (PEP) in the C 4 cycle (10). PPDK also synthesizes PEP in P. shermanii, Acetobacter xylinum, Rhodospirillum rubrum, Microbispora rosea (9), and Rhizobium meliloti (11). However, in E. histolytica, G. lamblia, C 3 plants, and trypanosomatids the role of PPDK has not been determined.In the reaction catalyzed by PPDK the transfer of the phosphoryl groups from PEP and PP i to AMP is mediated by a catalytic histidine (His) residue (12). This residue rotates 45 Å from the PEP/Pyr domain to the AMP-ATP/PP i -P i domain after its phosphorylation by PEP (12) and is also phosphorylated by a phosphoryl group derived from PP i (13). Enzymes that have function...

show abstract

Entamoeba histolytica: kinetic and molecular evidence of a previously unidentified pyruvate kinase

Cited by 19 publications

References 45 publications

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

Kinetic Mechanism and Metabolic Role of Pyruvate Phosphate Dikinase from Entamoeba histolytica

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