Mechanism of succinate production
Procyclic trypanosomes oxidise not only carbohydrates but also fatty acids and amino acids, such as proline, to carbon dioxide and water indicating the presence of a functional mitochondrial citric-acid cycle. Most of the enzymes of the cycle - citrate synthase (Durieux et al., 1991), aconitase (Overath et al., 1986), isocitrate dehydrogenase (Durieux et al., 1991; Opperdoes et al., 1977), succinate thiokinase (Jenkins et al., 1988), succinate dehydrogenase (SDH, Opperdoes et al., 1981; Durieux et al., 1991), fumarase (Durieux et al., 1991), malate dehydrogenase (Opperdoes and Cottem, 1982; Uttaro and Opperdoes, unpublished) - have all been detected in procyclic forms of T. brucei. Despite the functionality of this cycle, the insect stages secrete into the extracellular medium, in addition to carbon dioxide, large amounts of succinate and acetate, as well as some alanine (see above). Apparently, the capacity of the cycle is limited so that it is not able to cope with all the carbon units coming from glycolysis, from fatty acids and from amino-acid oxidation. The formation of succinate has been interpreted by some authors as an indication for the presence of an active fumarate reductase (FR) and indeed the enzyme has been described for the procyclic trypomastigote (Mracek et al 1991; Turrens, 1989; Turrens et al., 1996). However, other authors have described the presence of a succinate dehydrogenase (Opperdoes et al., 1981; Durieux et al., 1991). It is unlikely that the cycle runs in both directions simultaneously since in anaerobic bacteria and in helminths and Crustaceae, where (part of) the cycle runs in the reductive direction, different enzymes are required. In this respect it is interesting to note that the ATP-dependent succinate thiokinase, or succinyl CoA synthase, that is thought to function in the direction of succinyl CoA breakdown, and not the GTP-dependent succinate thiokinase thought to catalyse the reverse reaction, drastically increases with transformation form bloodstream form to insect stage (Jenkins et al., 1988). Moreover, in aerobic organisms SDH transfers its reducing equivalents via ubiquinone to the mitochondrial respiratory chain and ubiquinone Q9 is present in the trypanosomatids. FR uses either menaquinone (in prokaryotes), or rhodoquinone (in eukaryotes), rather than ubiquinone (Van Hellemond et al., 1995). Neither menaquinone, nor rhodoquinone have been detected in trypanosomatids (Tielens, personal communication). So most likely, the different authors that have measured the activity of FR or SDH have measured one and the same enzyme - SDH - but assayed in two different directions.