Glycolysis in Entamoeba histolytica. Biochemical characterization of recombinant glycolytic enzymes and flux control analysis

The synthesis of ATP in the human parasite Entamoeba histolytica is carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose-6-phosphate isomerase, inorganic pyrophosphate-dependent phosphofructokinase, fructose-1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes from E. histolytica. Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. An in vitro reconstruction of the final stages of glycolysis was made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH.     

Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of a single GlcNAc to the Ser or Thr of nucleocytoplasmic proteins. OGT activity, which may compete with that of kinases, is involved in signaling in animals and plants, and abnormalities in OGT activities have been associated with type 2 diabetes. Here, we show that ogt genes that predict enzymes with characteristic tetratricopeptide repeats and a spindly domain are present in some protists (Giardia, Cryptosporidium, Toxoplasma, and Dictyostelium) but are absent from the majority of protists examined (e.g., Plasmodium, Trypanosoma, Entamoeba, and Trichomonas). Similarly, ogt genes are present in some fungi but are absent from numerous other fungi, suggesting that secondary loss is an important contributor to the evolution of ogt genes. Nucleocytosolic extracts of Giardia and Cryptosporidium show OGT activity, and recombinant Giardia and Cryptosporidium OGTs are active in yeast and bacteria, respectively. These results suggest the possibility that O-GlcNAc modification of nucleocytosolic proteins also has function(s) in simple eukaryotes.     

Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis

Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.     

Pyruvate kinase isozymes: Ancient diversity retained in modern plant cells

Pyruvate kinase is a key regulatory enzyme of glycolysis. Phylogenetic analyses of the sequences coding for pyruvate kinase from plants and other organisms revealed unexpected diversity of this glycolytic enzyme in the progenitor of the present-day eukaryotes. Plants contain an ancient lineage of cytosolic pyruvate kinase, which may have diverged from the animal pyruvate kinase genes prior to the plant-animal divergence. The plant cytosolic pyruvate kinase genes are no more closely related to the animal and fungal pyruvate kinase genes than to the prokaryotic pyruvate kinase genes. The results suggest that the plant pyruvate kinase genes and the animal-fungal pyruvate kinase genes have descended from divergent isozymes which existed in the progenitor of the present-day eukaryotes and prokaryotes.     

Reconstructing the mosaic glycolytic pathway of the anaerobic eukaryote Monocercomonoides

All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes; Monocercomonoides, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of Monocercomonoides and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of Eukaryota, Bacteria, and Archaea. We established that, firstly, Monocercomonoides possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.     

Programmed cell death in Giardia

Programmed cell death (PCD) has been observed in many unicellular eukaryotes; however, in very few cases have the pathways been described. Recently the early divergent amitochondrial eukaryote Giardia has been included in this group. In this paper we investigate the processes of PCD in Giardia. We performed a bioinformatics survey of Giardia genomes to identify genes associated with PCD alongside traditional methods for studying apoptosis and autophagy. Analysis of Giardia genomes failed to highlight any genes involved in apoptotic-like PCD; however, we were able to induce apoptotic-like morphological changes in response to oxidative stress (H2O2) and drugs (metronidazole). In addition we did not detect caspase activity in induced cells. Interestingly, we did observe changes resembling autophagy when cells were starved (staining with MDC) and genome analysis revealed some key genes associated with autophagy such as TOR, ATG1 and ATG 16. In organisms such as Trichomonas vaginalis, Entamoeba histolytica and Blastocystis similar observations have been made but no genes have been identified. We propose that Giardia possess a pathway of autophagy and a form of apoptosis very different from the classical known mechanism; this may represent an early form of programmed cell death.     

The biochemical properties and phylogenies of phosphofructokinases from extremophiles

The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.     

Iron hydrogenases and the evolution of anaerobic eukaryotes

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis: a free-radical enzyme system conserved across divergent eukaryotic lineages

Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified.     

Regulation of alcoholic fermentation in coleoptiles of two rice cultivars differing in tolerance to anoxia

To investigate regulation of anaerobic carbohydrate catabolism in anoxia-tolerant plant tissue, rate of alcoholic fermentation and maximum catalytic activities of four key enzymes were assessed in coleoptiles of two rice cultivars that differ in tolerance to anoxia. The enzymes were ATP-dependent phosphofructokinase (PFK), pyrophosphate-dependent phosphofructokinase (PFP), pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH). During anoxia, rates of coleoptile elongation and ethanol synthesis were faster in the more tolerant variety Calrose than in IR22. Calrose coleoptiles, in contrast to IR22, also showed a sustained Pasteur effect, with the estimated rate of glycolysis during anoxia being 1.4-1.7-fold faster than that of aerobic coleoptiles. In Calrose after 5 d anoxia, maximum catalytic activities of crude enzyme extracts were (in mumol substrate g-1 fresh weight min.-1) 170-240 for ADH, 4-6 for PDC and PFP and 0.4-0.7 for PFK. During anoxia, activity per coleoptile of all four enzymes increased 3-5.5-fold, suggesting that PFK, and PFP, like PDC and ADH, are synthesised in anoxic rice coleoptiles. Enzyme activities, on a fresh weight basis, were lower in IR22 than in Calrose. In vivo activities of PDC and PFK in anoxic coleoptiles from both cultivars were calculated using in vitro activities, estimated substrate levels, cytoplasmic pH, and S0.5 (the substrate level at which 0.5Vmax is reached, without inferring Michaelis-Menten kinetics). Data indicated that potential carbon flux through PFK, rather than through PDC, more closely approximated rates of alcoholic fermentation. That PFK is an important site of regulation was supported further for Calrose coleoptiles by a decrease in the concentration of its substrate pool (F-6-P + G-6-P) following the onset of anoxia. By contrast, in IR22, there was little evidence for control by PFK, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.     

Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.     

Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis

Two genes coding for Trichomonas vaginalis glucokinase were isolated and sequenced. The putative translation products have molecular masses of 41,584 and 41,772 Da, corresponding to 375 and 377 amino acids, respectively. These values agree with data determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the enzyme purified from the organism. The two sequences showed 78% amino acid identity. The sequences and their phylogenetic reconstruction show that they are members of a glucokinase/fructokinase protein family found in eubacteria and also in the eukaryote Giardia lamblia and are only distantly related to typical eukaryotic hexokinases. The results indicate that the evolutionary past of this enzyme, catalyzing the first step of glycolysis in T. vaginalis, is different from that of the enzyme performing this key role in almost all other eukaryotes.     

Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?

Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.     

Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships

Sequences of putative fructose-1,6-bisphospate aldolases (FBA) in five amitochondriate unicellular eukaryotes, the diplomonads Giardia intestinalis (published earlier) and Spironucleus barkhanus, the pelobiont Mastigamoeba balamuthi,the entamoebid Entamoeba histolytica, and the parabasalid Trichomonas vaginalis all belong to Class II of FBAs and are highly similar to each other (>48% amino acid identity). The five protist sequences, however, do not form a monophyletic group. Diplomonad FBAs share a most recent common ancestor, while FBAs of the three other protist species are part of a lineage that also includes sequences from a few eubacteria (Clostridium difficile, Treponema pallidum, Chlorobium tepidum). Both clades are part of the Type B of Class II aldolases, a complex that contains at least three additional lineages (subgroups) of enzymes. Type B enzymes are distant from Type A Class II aldolases, which consists of a number of bacterial and fungal enzymes and also contains the cytosolic FBA of Euglena gracilis. Class II aldolases are not homologous to Class I enzymes, to which animal and plant enzymes belong. The results indicate that amitochondriate protists acquired their FBAs from separate and different sources, involving lateral gene transfer from eubacteria, than did all other eukaryotes studied so far and underscore the complex composition of the glycolytic machinery in unicellular eukaryotes.     

WhatEntamoeba histolytica andGiardia lamblia tell us about the evolution of eukaryotic diversity

Entamoeba histolytica andGiardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.     

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphate-dependent (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.