Barbara M. Bakker

Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes.

In trypanosomes the first part of glycolysis takes place in specialized microbodies, the glycosomes. Most glycolytic enzymes of Trypanosoma brucei have been purified and characterized kinetically. In this paper a mathematical model of glycolysis in the bloodstream form of this organism is developed on the basis of all available kinetic data. The fluxes and the cytosolic metabolite concentrations as predicted by the model were in accordance with available data as measured in non-growing trypanosomes, both under aerobic and under anaerobic conditions. The model also reproduced the inhibition of anaerobic glycolysis by glycerol, although the amount of glycerol needed to inhibit glycolysis completely was lower than experimentally determined. At low extracellular glucose concentrations the intracellular glucose concentration remained very low, and only at 5 mM of extracellular glucose, free glucose started to accumulate intracellularly, in close agreement with experimental observations. This biphasic relation could be related to the large difference between the affinities of the glucose transporter and hexokinase for intracellular glucose. The calculated intraglycosomal metabolite concentrations demonstrated that enzymes that have been shown to be near-equilibrium in the cytosol must work far from equilibrium in the glycosome in order to maintain the high glycolytic flux in the latter.

What Controls Glycolysis in Bloodstream Form Trypanosoma brucei

On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream formTrypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhütter, H.-G. (1995) Eur. J. Biochem. 229, 403–418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these “irreversible” enzymes mediocre drug targets.

In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria.

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.

Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei

Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved.

Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs.

Glycolysis is the only ATP-generating process in bloodstream form trypanosomes and is therefore a promising drug target. Inhibitors which decrease significantly the glycolytic flux will kill the parasites. Both computer simulation and experimental studies of glycolysis in bloodstream form Trypanosoma brucei indicated that the control of the glycolytic flux is shared by several steps in the pathway. The results of these analyses provide quantitative information about the prospects of decreasing the flux by inhibition of any individual enzyme. The plasma membrane glucose transporter appears the most promising target from this perspective, followed by aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. Non-competitive or irreversible inhibitors would be most effective, but it is argued that potent competitive inhibitors can be suitable, provided that the concentration of the competing substrate cannot increase unrestrictedly. Such is the case for inhibitors that compete with coenzymes or with blood glucose.

Yeast cells with a specific cellular make-up and an environment that removes acetaldehyde are prone to sustained glycolytic oscillations.

Glycolytic oscillations can be induced by adding glucose to starved Saccharomyces cerevisiae cells and, after a steady state has been established, cyanide. Transient oscillations or limit‐cycle oscillations can be induced depending on the growth phase in which the cells are harvested. To find what causes these differences in the dynamic behaviour, we analyzed glycolytic enzyme activities at different growth phases. The hexokinase activity increased by a factor of three after growth substrate transition from glucose to ethanol; the other measured activities remained constant. Cyanide was found not only to block respiration, but also to trap acetaldehyde. Both cyanide actions appear necessary for the occurrence of sustained glycolytic oscillations.

Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae

The tendency of Saccharomyces cerevisiae to favor alcoholic fermentation over respiration is a complication in aerobic, biomass-directed applications of this yeast. Overproduction of Hap4p, a positive transcriptional regulator of genes involved in respiratory metabolism, has been reported to positively affect the balance between respiration and fermentation in aerobic glucose-grown batch cultures. In this study, the effects of HAP4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN.PK 113-7D under a variety of growth conditions. In aerobic glucose-limited chemostat cultures, overexpression of HAP4 increased the specific growth rate at which aerobic fermentation set in by about 10% relative to the isogenic wild-type. Upon relief of glucose-limited conditions, the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain. The effect of Hap4p overproduction was most drastic in aerobic, glucose-grown chemostat cultures in which ammonium was limiting. In such cultures, the biomass yield on glucose was double that of the wild-type.

Regulation and Control of Compartmentalized Glycolysis in Bloodstream Form Trypanosoma brucei

Unlike other eukaryotic cells, trypanosomes possess a compartmentalized glycolytic pathway. The conversion of glucose into 3-phosphoglycerate takes place in specialized peroxisomes, called glycosomes. Further conversion of this intermediate into pyruvate occurs in the cytosol. Due to this compartmentation, many regulatory mechanisms operating in other cell types cannot work in trypanosomes. This is reflected by the insensitivity of the glycosomal enzymes to compounds that act as activity regulators in other cell types. Several speculations have been raised about the function of compartmentation of glycolysis in trypanosomes. We calculate that even in a noncompartmentalized trypanosome the flux through glycolysis should not be limited by diffusion. Therefore, the sequestration of glycolytic enzymes in an organelle may not serve to overcome a diffusion limitation. We also search the available data for a possible relation between compartmentation and the distribution of control of the glycolytic flux among the glycolytic enzymes. Under physiological conditions, the rate of glycolytic ATP production in the bloodstream form of the parasite is possibly controlled by the oxygen tension, but not by the glucose concentration. Within the framework of Metabolic Control Analysis, we discuss evidence that glucose transport, although it does not qualify as the sole rate-limiting step, does have a high flux control coefficient. This, however, does not distinguish trypanosomes from other eukaryotic cell types without glycosomes.

Using metabolic control analysis to improve the selectivity and effectiviness of drugs against parasitic diseases.

Trypanosoma brucei is the parasite that causes African sleeping sickness in humans and the related disease nagana in cattle. The development of drugs is hampered by the many similarities between this parasite and the cells of its host. Until now advanced drug-design strategies have focussed on the differences between the three-dimensional structure of trypanosome and human enzymes (Verlinde & Hol, 1994). We propose that the selectivity of a drug can be further enhanced by choosing a target enzyme with a high flux control coefficient in the parasite and a low flux control coefficient in the host cells. Trypanosome glycolysis is a very suitable model system for this approach. In the mammalian bloodstream T. brucei depends completely on glycolysis (Michels, 1988). In many of its host cells glycolysis is also essential. The organization of this pathway and the regulation of its enzymes differ greatly between the two organisms (Michels, 1988). Most conspicuously, in trypanosomes part of glycolysis takes place in specialized organelles (glycosomes), whereas in the host the corresponding pathway is localized in the cytosol. In view of this and other differ-ences there is a fair chance that the distributrion of the control of the glycolytic flux over the glycolytic enzymes will also be different.

Hierarchies in control

The living cell functions by virtue of an enormous number of different processes. It is one of the most difficult challenges of modern biology to elucidate how all those processes are coordinated quantitatively so as to lead to a viable system with optimal responses to various changes in the environment. The biochemical and biophysical processes of the living cell do not constitute a network with random connections. In this paper we shall discuss that cell function is organized in hierarchical substructures. We will briefly touch on the topics of (i) metabolic control and regulated gene expression, (ii) time dependent metabolism in intact yeast cells, and (iii) metabolite channelling.