Glycolysis

Glycolysis (glycose+lysis= breaking of glucose) is a sequence of 10 definite reactions which leads to breaking of glucose into pyruvate. This is the first of four steps of aerobic respiration. This cytosolic metabolic pathway produces a net gain of 2 ATPs. This step is also common to anaerobic respiration as it does not require O2. In anaerobic respiration, pyruvate will then be converted to lactate (e.g. skeletal musles) or ethanol and carbon dioxide (e.g. yeast) to re-oxidise electron carrier NADH to provide NAD+ for glycolysis.

 

The apicomplexan Toxoplasma gondii possesses all the ten enzymes of glycolysis in the genome. It also possesses 2 lactate dehydrogenase isoforms (converts pyruvate to lactate). The lactate dehydrogenases are expressed in stage specific manner, LDH1 is tachyzoite specific and LDH2 is bradyzoite specific [1, 2, 3]. These LDH isoforms also showed differences in substrate specificities between them and in comparison to Plasmodium LDH. They also showed differences in sensitivities to LDH inhibitors such as gossypol [4, 5]. In addition, it has been mentioned that the comparison of X-ray crystal structure of LDH1 with human muscle and heart-specific isoforms shows significant differences in residues in active site. This suggests it will make a good drug target for encephalitis and other complications of Toxoplasmosis in immuno-compromised patients [5]. The absence of two subunits of ATP synthase and the presence of lactate dehydrogenase in P. falciparum suggests that it relies mainly on glycolysis for ATP generation. It has also been demonstrated in intra-erythrocytic stage P. falciparum that all the glucose consumed are converted to fermentation products [6]. Although electron transport chain is functional and essential for ATP production at least in tachyzoite [7], it is not exactly known of the role of different metabolic pathways in ATP generation in T. gondii. The glycolysis and anaerobic fermentation may also be the main source of ATP in other life cycle stages.

 

The enzymes NAD-dependent glycerol-3-phosphate dehydrogenase and FAD-dependent glycerol-3-phophate dehydrogenase which catalyse bidirectional conversion of glycerol-3-phosphate to glycerone phosphate were added to glycolysis pathway in MPMP. This is because the reaction catalysed by these  enzymes will lead to increased glycolytic intermediate glycerone phosphate, which will increase pyruvate and ATP production via the actions of triose phosphate isomerise and enzymes downstream of it. The above mentioned enzymes and other enzymes added in MPMP glycolysis pathway such as aldehyde reductase, phosphoglucomutase and acylphosphatase are also present in T. gondii and added to the pathway here. In addition, gluconeogenesis enzyme, fructose bisphosphatase (absent in P. falciparum) is present in T. gondii and added to the glycolysis pathway. The enzyme glycerol kinase, present in P. falciparum glycolysis pathway is absent in Toxoplasma genome. 

 

The three dimensional models and phylogeny analysis showed that the enzymes enolase and glucose-6-phosphate isomerise are related to plant homologues rather than animal enzymes suggesting that they can also be possible drug targets [8]. The enolase isoform ENO2 is tachyzoite specific and ENO1 is bradyzoite specific. The labelling experiments showed more intense staining of both isoforms in nucleus compared to cytosol in proliferating phases of all life cycle stages. When compared to cytosol, weaker nuclear staining was observed in mature phases suggesting the nuclear role of enolase in regulation of transcription/cell division in addition to its role in glycolysis [3]. The same has also been observed with P. falciparum enolase, which also localises to nucleus, vacuole and merozoite surface in addition to cytosol [9].

 

The pyrophosphate-dependent activity of phosphofructokinase enzyme was observed in three Apicomplexa parasites, T. gondii, Eimeria tenella and Cryptosporidium parvum in the early nineties. The Toxoplasma enzyme was identified and purified to near homogeneity from extracts and the enzymes assays were carried out identify substrate specificities and kinetic properties [10]. This enzyme catalyses the reaction in both forward and reverse directions with pyrophosphate as co-substrate. None of the nucleoside phosphates including ATP was able to act as either phosphate donor or effector for the enzyme. The enzyme activity is dependent on Mg(2+) ions. This pyrophosphate specific enzyme was also characterised in other apicomplexans Eimeria [11] and Cryptosporidium [12]. All showed activity with pyrophosphate and not with ATP. These protozoan enzymes are homodimers and are not allosterically regulated.

 

Protein EC Number Gene id Protein localisation Localisation data source
Aldehyde reductase 1.1.1.21 TGME49_208040    
Lactate dehydrogenase (LDH1) 1.1.1.27 TGME49_232350 Cytosol; Inner membrane complex Apiloc; Previous publication
Lactate dehydrogenase (LDH2) 1.1.1.27 TGME49_291040 Cytosol; Mitochondria Apiloc; Previous publication
Glycerol-3-phosphate dehydrogenase 1.1.1.8 TGME49_307570 Mitochondrion Previous publication
Glycerol-3-phosphate dehydrogenase 1.1.1.8 TGME49_210260    
Glycerol-3-phosphate dehydrogenase 1.1.5.3 TGME49_263730 Mitochondrion Previous publication
Glyceraldehyde 3-P dehydrogenase 1.2.1.12 TGME49_269190 Endoplasmic reticulum; Mitochondrion Apiloc; Previous publication
Glyceraldehyde 3-P dehydrogenase 1.2.1.12 TGME49_289690 Cytosol; Inner membrane complex; Mitochondrion Apiloc; Previous publication
Hexokinase 2.7.1.1 TGME49_265450 Cytosol Apiloc; Previous publication
Phosphofructokinase 2.7.1.90 TGME49_226960 Cytosol Apiloc; Previous publication
Phosphofructokinase 2.7.1.90 TGME49_240890 Mitochondrion Previous publication
Phosphofructokinase 2.7.1.90 TGME49_281400 Cytosol Previous publication
Pyruvate kinase 2.7.1.40 TGME49_256760 Cytosol; Inner membrane complex; Apicoplast Apiloc; Previous publication
Phosphoglycerate kinase 2.7.2.3 TGME49_222020 Apicoplast; Nucleus Apiloc; Previous publication
Phosphoglycerate kinase 2.7.2.3 TGME49_318230 Cytosol Apiloc; Previous publication
Fructose bisphosphatase 3.1.3.11 TGME49_247510 Cytosol Apiloc; Previous publication
Fructose bisphosphatase 3.1.3.11 TGME49_205380 Cytosol Apiloc
Acylphosphatase 3.6.1.7 TGME49_258940    
Aldolase 4.1.2.13 TGME49_236040 Cytosol; Inner membrane complex; Golgi Apiloc; Previous publication
Aldolase 4.1.2.13 TGME49_236050    
Aldolase 4.1.2.13

TGME49_321900

(release 8 gene model)

   
Enolase (ENO2) 4.2.1.11 TGME49_268850 Nucleus; Cytosol; Mitochondrion? Apiloc; Previous publication
Enolase (ENO1) 4.2.1.11 TGME49_268860 Nucleus; Cytosol Apiloc; Previous publication
Triose phosphate isomerase 5.3.1.1 TGME49_225930 Cytosol Apiloc; Previous publication
Phosphoglucose isomerase 5.3.1.9 TGME49_283780 Cytosol Apiloc; Previous publication
Phosphoglycerate mutase 5.4.2.1 TGME49_222910 Mitochondrion Previous publication
Phosphoglycerate mutase 5.4.2.1 TGME49_273030    
Phosphoglycerate mutase 5.4.2.1 TGME49_297060 Cytosol Apiloc; Previous publication
Phosphoglycerate mutase 5.4.2.1 TGME49_318190 Cytosol Previous publication
Phosphoglucomutase 5.4.2.2 TGME49_285980 Cytosol; Apical Apiloc; Previous publication
Phosphoglucomutase 5.4.2.2 TGME49_318580 Cytosol; Mitochondrion Apiloc; Previous publication
Monocarboxylate transporter none TGME49_233540    
Monocarboxylate transporter none TGME49_297245    
Facilitative glucose transporter (Hexose transporter) none TGME49_214320 Plasma membrane Apiloc; GO annotation

 

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Sources and fates of metabolites

 

Substrate Source pathways Product Fate pathways
alpha-D-Glucose Host, Starch and galactose metabolism alpha-D-Glucose-6P Pentose phosphate cycle, Starch and galactose metabolism
    alpha-D-Glucose-1P Starch and galactose metabolism, Pyrimidine metabolism
beta-D-Fructose-6P Pentose phosphate cycle beta-D-Fructose-6P Pentose phosphate cycle, Aminosugars metabolism, Starch and galactose metabolism
Glyceraldehyde-3P Pentose phosphate cycle Glyceraldehyde-3P Pentose phosphate cycle, Pyridoxal phosphate (vitamin B6) metabolism
    Phosphoenolpyruvate Pyruvate metabolism, Shikimate metabolism
    Pyruvate Pyruvate metabolism, Alanine metabolism
    sn-glycerol-3P Phosphatidylethanolamine and phosphatidylserine metabolism
    Glycerone-P Fatty acid synthesis in the apicoplast
    Lactate Host