Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, beta-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.

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A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.Trypanosomatid parasites (Trypanosoma and Leishmania) are responsible for serious diseases of mankind in tropical and subtropical countries worldwide. These diseases affect millions of people, and hundreds of millions are at risk of becoming infected. Unfortunately, available treatments are largely inadequate. Currently used drugs are inefficacious and toxic. There is a desperate need for new effective and safe drugs, particularly in view of the development and spreading of drug resistance (1).Glycolysis plays an important role in the energy metabolism of these protozoan organisms, notably of Trypanosoma brucei when it lives in the blood of its mammalian host, causing a disease called sleeping sickness or human African trypanosomiasis in man and nagana in cattle (2, 3). This bloodstream form of T. brucei is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply. Oxidative metabolism involving mitochondrial Krebs cycle enzymes and oxidative phosphorylation are largely repressed. Therefore, glycolysis has been perceived as a potentially good target for anti-trypanosome drugs. Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner: the majority of the glycolytic enzymes are sequester...

Trypanosomatid parasites cause serious diseases among humans, livestock, and plants. They belong to the order of the Kinetoplastida and form, together with the Euglenida, the phylum Euglenozoa. Euglenoid algae possess plastids capable of photosynthesis, but plastids are unknown in trypanosomatids. Here we present molecular evidence that trypanosomatids possessed a plastid at some point in their evolutionary history. Extant trypanosomatid parasites, such as Trypanosoma and Leishmania, contain several ''plant-like'' genes encoding homologs of proteins found in either chloroplasts or the cytosol of plants and algae. The data suggest that kinetoplastids and euglenoids acquired plastids by endosymbiosis before their divergence and that the former lineage subsequently lost the organelle but retained numerous genes. Several of the proteins encoded by these genes are now, in the parasites, found inside highly specialized peroxisomes, called glycosomes, absent from all other eukaryotes, including euglenoids.

Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 o rigid body rocking motion of the A-and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.Allosteric regulation ("the second secret of life" quotation ascribed to Jacob Monod) (see Ref. 1) controls many important cellular processes, including signal transduction, transcription, and metabolism (2). It describes the effect of binding one ligand on the subsequent binding of a second ligand at a topographically distinct site. Human pyruvate kinase (hPYK) 2 provides a striking example of the significance of allosteric regulation: a splicing switch of the primary RNA transcript to yield the M1 or M2 isoenzymes is now known to be responsible for the Warburg effect in cancer (3, 4) and opens up the possibility of developing isoform specific therapeutics (5). Additional mechanisms of activity regulation, including the binding of amino acids, phosphorylation, and the binding of oncoproteins, may provide further therapeutic approaches for targeting hPYK (6, 7).Most allosterically regulated proteins are enzymes in which the binding of an activator or inhibitor to the effector site can affect the binding of a substrate at the active site. The MonodWyman-Changeux model of allostery (8) suggests that oligomeric enzymes undergo symmetrical transitions (classically between the T-and R-states) 3 (36) that can be stabilized by ligand binding. However, there are now examples of allosteric control that do not show obvious conformational change (9), and a growing body of work exists to support the idea that flexible regions of molecules (which exist as an ensemble of conformers) may undergo allosteric regulation by changes in the conformer population (10). There are examples of allosteric enzymes in which the same protein has been captured in both a T-state (which has low affinity for substrate) and an R-state (which has higher affinity for substrate), and some structural insight has been obtained by the study of at least one of the allosteric states of Ͼ50 proteins (10 -12). However, a full understanding of the allosteric effect requires structural information on each of four states: (i) apoenzyme, (ii) active site complex, (iii) eff...

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The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease, is reported. The final model at 2.8 A î includes the bound cofactor NAD + and 90 water molecules per monomer and resulted in an R ftor of 20.1%, R free = 22.3%, with good geometry indicators. The structure has no ions bound at the active site resulting in a large change in the side chain conformation of Arg PRW which as a consequence forms a salt bridge to Asp PIH in the present structure. We propose that this conformational change could be important for the reaction mechanism and possibly a common feature of many GAPDH structures. Comparison with the human enzyme indicates that interfering with this salt bridge could be a new approach to specific inhibitor design, as the equivalent to Asp PIH is a leucine in the mammalian enzymes. z 1998 Federation of European Biochemical Societies.

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