The Embden-Meyerhof-Parnass (EMP) pathway of glycolysis
The long slender bloodstream form
The vertebrate stage of the African trypanosome dwells in the blood and tissue fluids of its mammalian host, where it has access to an unlimited source of glucose which is maintained at the relatively constant concentration of 5 mM. Because it is so abundant, glucose serves essentially as the sole source of carbon and energy for the trypanosome. It is metabolised at a rate exceeding that found in most other eukaryotic cells, but despite this fact the bloodstream-form trypanosome utilises only a relatively small portion of its total protein for this activity. This is explained by the fact that where in most cells the glycolytic pathway takes place in the cytosol, in the trypanosome the first seven enzymes are localized within a membrane-bound organelle (Opperdoes and Borst, 1977; Opperdoes, 1987). Although this organelle resembles the microbody or peroxisome of other cell types, it lacks typical peroxisomal enzymes such as catalase and acylCoA oxidase, while 90% of its protein is glycolytic enzyme (Misset et al., 1986). Therefore, these highly specialised microbodies have been called "glycosomes" (Opperdoes and Borst, 1977). Because neither a functional citric-acid cycle nor a classical mitochondrial respiratory chain are present, pyruvate, the end-product of the glycolytic pathway, is secreted as such into the host's bloodstream and is not metabolised to lactate or carbon dioxide plus water, as in most other organisms. Hence, little ATP is produced and this is done by substrate-level phosphorylation at the level of the phosphoglycerate kinase and pyruvate kinase steps.
Because bloodstream trypanosomes are completely dependent on glycolysis for their supply of ATP and because the organisation of their glycolytic pathway is very different from that in the host, glycolysis has been identified as a promising target for the development of new drugs against African sleeping sickness (Michels, 1988). For this reason the pathways of carbohydrate metabolism in the vertebrate stage of T. brucei have been studied in great detail.
In the long slender bloodstream form the EMP is solely responsible for the oxidation of glucose to pyruvate, with the concomitant generation of 2 moles of ATP per mole of glucose consumed (Fig. 1). Glucose is the preferred energy source for the bloodstream form, but fructose and mannose can also be metabolised. Although probably not of physiological relevance, glycerol may also be used as an energy substrate, at a rate equal or even higher than that of glucose consumption. The ATP yield in this case is only half that of glucose, which explains why the rate of respiration with this substrate sometimes supersedes that observed with glucose. Due to the absence of a mitochondrial pyruvate dehydrogenase complex, the long slender form does not oxidise glucose beyond pyruvate and excretes it as such into the bloodstream. Since a functional citric-acid cycle and mitochondrial respiratory chain are absent, amino acids and fatty acids, cannot be not utilized as energy substrates by these life-cycle stages. Moreover, bloodstream forms contain neither any carbohydrate stores, such as glycogen or other polysaccharides, nor any energy reserves of any significance, such as creatine phosphate or polyphosphates. Thus depletion of trypanosomes of an energy substrate such as glucose will result in a rapid drop of cellular ATP levels and a total loss of motility.
Figure 1 (Glycolysis of the bloodstream form here )
The bloodstream form of T. brucei carries out an aerobic type of glycolysis. This means that the NADH that is produced in the glycosome by the glyceraldehyde-phosphate dehydrogenase reaction is reoxidized indirectly by molecular oxygen. For this oxidation the reducing equivalents have to cross the glycosomal membrane in order to reach the terminal oxidase located in the mitochondrial inner membrane. The African trypanosome transfers the reducing equivalents coming from NADH by a glycerol-3-phosphate (G3P) : dihydroxyacetone-phosphate (DHAP) shuttle. This shuttle comprises two reactions, the reduction of DHAP to G3P by the glycosomal NAD-linked G3P dehydrogenase and the reversed reaction that is catalysed by a FAD-linked G3P dehydrogenase in the mitochondrial inner membrane. By analogy with the mammalian G3P dehydrogenases, this enzyme is most likely located at the outside of the mitochondrial inner membrane, so that the G3P does not have to cross this membrane in order to be oxidised. The latter dehydrogenase transfers the reducing equivalents to ubiquinone (UQ9) which is then reduced to ubiquinol. The above shuttle can only be operational when the glycosomal membrane is permeable to G3P and DHAP. However, latency measurements carried out with intact glycosomes have shown that the glycosomal membrane constitutes a diffusion barrier for the glycolytic intermediates. Therefore, Opperdoes and Borst (1977) have proposed a specific DHAP : G3P antiport allowing for the entry of 1 molecule of DHAP for each molecule of G3P that leaves the glycosome. Although, so far, no direct experimental evidence has been brought forward for the presence of such an antiport, recent mathematical modelling of the glycolytic pathway also required a DHAP : G3P antiport in the glycosomal membrane in order to explain the observed sensitivity of anaerobic glycolysis to inhibition by glycerol through mass action (Bakker et al., 1998).
Ubiquinol is oxidised by a ubiquinol : oxygen oxidoreductase present in the mitochondrial inner membrane. This enzyme, which is insensitive to cyanide, but is sensitive to salicyl hydroxamic acid (SHAM), resembles the so-called alternative oxidases that have been described for plants (Siedow and Umbach, 1995) and certain fungi such as Neurospora crassa (Lambowitz et al., 1989; Day et al., 1995). In neither fungi nor trypanosomes evidence has been found for an involvement of the enzyme in transmembrane proton translocation. The enzyme has also been detected in Phytomonas (Sanchez-Moreno et al., 1992; Van Hellemond, 1997), but has been reported to be absent from Leishmania, where neither the activity, nor the gene encoding the enzyme, could be detected (Van Hellemond, 1997). The mammalian host does not have such an alternative oxidase and transfers its electrons towards oxygen via a the classical respiratory chain containing the cytochromes b, c, c1 and aa3. Thus, the T. brucei alternative oxidase has been suggested as an interesting target for chemotherapy.
Inhibition of the trypanosome alternative oxidase by 1 mM of SHAM mimicks the effect of a lack of oxygen on the carbohydrate metabolism of the bloodstream form. Under these conditions, long slender bloodstream forms continue to utilize glucose at about the same rate as under aerobic conditions, but because the glycerol 3-phosphate : dihydroxyacetone phosphate cycle is now inoperative, glycerol 3-phosphate accumulates inside the glycosome, while the glycosomal ATP concentration rapidly drops (Opperdoes and Borst, 1977). This situation leads to a condition where a reversal of an essentially irreversible glycerol kinase reaction by mass action becomes possible, leading to the production of glycerol as an end-product of anaerobic glycolysis with the synthesis of 1 mole of ATP for each glycerol produced (Hammond et al., 1980 a,b). This compensates for the loss of one mole of ATP in the glycosomal phosphoglycerate kinase reaction, because now one mole of phosphoglycerate, rather than two, is produced per mole of glucose consumed. As a consequence glucose is dismutated into equimolar amounts of pyruvate and glycerol, with net synthesis of 1 molecule of ATP (Opperdoes and Borst, 1977; Fairlamb and Opperdoes, 1986; Opperdoes, 1987). Together with pyruvate and glycerol, trace amounts of alanine are excreted, probably as a result of a the transamination of pyruvate in the cytosol.
Under anaerobic conditions (or with SHAM) bloodstream forms survive and remain motile, while cellular ATP levels drop to about 50% (Opperdoes et al., 1976). Because glycerol 3-phosphate cannot be oxidized to DHAP without molecular oxygen, glycerol cannot serve as a substrate in the absence of oxygen, while glucose does. Modeling of the aerobic/anaerobic transition of glycolysis suggests that it takes place at very low (micromolar) oxygen concentrations, close to that of the Km of trypanosomal glycerol-3-phosphate oxidase. The anaerobic pathway appears to be almost completely inoperative at oxygen tensions in the range of those found in venous and arterial blood (Eisenthal and Panes, 1985).
Inhibition of respiration alone is not sufficient to kill the organism and the trypanosome is able to survive as long as glycerol does not accumulate in the medium (Fairlamb et al., 1977), but once above several millimolar, glycerol becomes toxic. Due to mass action the reversal of the glycerol kinase reaction now becomes inhibited and glycolysis comes to a complete stop (Fairlamb et al., 1977). Since trypanosomes lack energy stores such as creatine phosphate or polyphosphates, cellular ATP levels rapidly drop to zero, leading to their complete immobilisation. This observation has been exploited for the development of a rational treatment of experimental animals infected with either T. brucei, T. rhodesiense or T. vivax. A simultaneous administration of SHAM and glycerol leads to an almost immediate lysis and disappearance of parasites from the circulation (Clarkson and Brohn, 1976). Permanent cures, however, were only obtained at concentrations of the drugs that were toxic to the animals (Van der Meer and Versluys-Broers, 1979; 1986).
Many new and effective inhibitors of the trypanosome alternative oxidase have been described (Grady et al., 1986 a,b; 1993) of which ascofuranone was the most potent (Minagawa et al., 1997). But so far, none of these have been developed into an effective anti-trypanosome drug.
The trypanosome alternative oxidase gene has recently been cloned and sequenced (Chauduri and Hill, 1996). Its inferred amino-acid sequence shows that the enzyme is homologous to the alternative oxidases of plants and fungi and transformation of an Escherichia coli mutant, lacking a functional cytochrome c oxidase, with the alternative oxidase gene of the trypanosome was able to restore respiration in this bacterium (Chauduri and Hill, 1996).
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