ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation. Fig. S1]. In glycolysis, ATP is first invested in 2 phosphorylation reactions, catalyzed by hexokinase (HXK) and phosphofructokinase (PFK). Only further downstream in the pathway the ATP is regained and then a surplus of ATP is generated. In glycerol metabolism, ATP is invested in the reaction catalized by glycerol kinase (GK) (Fig. 1).In the absence of specific regulation of HXK and PFK, the ATP produced by glycolysis could boost the flux through these enzymes above the capacity of the enzymes downstream, and hexose phosphates [glucose 6-phosphate (Glc6P), fructose 6-phosphate (Fru6P) and fructose 1,6-phosphate (Fru1,6BP)] would accumulate to extreme levels. By analogy to the turbo engine (which uses engine exhaust to boost performance), this property was called the ''turbo-design''of glycolysis (2). Many organisms avoid the negative side effects of the autocatalytic design of glycolysis by a tight feedback regulation of HXK and PFK, e.g., inhibition of HXK by Glc6P (3) or trehalose 6-phosphate (5). In yeast, deletion of trehalose-6-phosphate synthase leads to glucose toxicity, accumulation of hexose phosphates and rapid consumption of ATP (4). This ''turbo-explosion'' phenotype is rescued by reducing the expression of HXK (5) or glucose transporters (6).Trypanosoma brucei, the tropical parasite that causes the deadly African sleeping sickness, lacks feedback regulation of HXK and PFK (7-9). The parasite has a complex life cycle that alternates between insect and mammalian hosts; in the latter it lives in the bloodstream, supplied with a ready source of glucose. How then are trypanosomes protected against a possible turboexplosion of glycolysis?A key feature of trypanosome glycolysis is the compartmentation of the first 7 enzymes of glycolysis and 2 involved in glycerol metabolism inside peroxisome-like organelles called glycosomes (10). In the glycosome, ATP and redox levels are ba...
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, glucoselimited chemostat cultures of a gut2⌬ 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 nde1⌬ nde2⌬ 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 nde1⌬ nde2⌬ gut2⌬ 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 nde1⌬ nde2⌬ gut2⌬ 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.As in other eukaryotes, respiratory dissimilation of sugars by Saccharomyces cerevisiae leads to the reduction of NAD ϩ to NADH in separate cellular compartments. Cytosolic NADH is produced by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, as well as in assimilatory reactions (1, 30). In the mitochondrial matrix, NADH is formed by the tricarboxylic acid cycle and the pyruvate-dehydrogenase complex. Under anaerobic conditions, glucose dissimilation occurs exclusively via alcoholic fermentation, which is a redox-neutral process. The additional NADH originating from biomass production can be reoxidized via glycerol production (30). Under aerobic conditions, glycerol production is not necessary, because the reoxidation of cytosolic NADH can be coupled to the mitochondrial respiratory chain.Although the outer mitochondrial membrane is permeable to NADH (17), the inner membrane is not (35). Therefore, coupling of NADH reoxidation to the respiratory chain has to occur on both sides of the mitochondrial inner membrane. In plant mitochondria, cytosolic NADH can be oxidized either by an external NADH dehydrogenase or by a redox shuttle (14), whereas in the mitochondrial mat...
An important question is to what extent metabolic fluxes are regulated by gene expression or by metabolic regulation. There are two distinct aspects to this question: (i) the local regulation of the fluxes through the individual steps in the pathway and (ii) the influence of such local regulation on the pathway's flux. We developed regulation analysis so as to address the former aspect for all steps in a pathway. We demonstrate the method for the issue of how Saccharomyces cerevisiae regulates the fluxes through its individual glycolytic and fermentative enzymes when confronted with nutrient starvation. Regulation was dissected quantitatively into (i) changes in maximum enzyme activity (V max, called hierarchical regulation) and (ii) changes in the interaction of the enzyme with the rest of metabolism (called metabolic regulation). Within a single pathway, the regulation of the fluxes through individual steps varied from fully hierarchical to exclusively metabolic. Existing paradigms of flux regulation (such as single-and multisite modulation and exclusively metabolic regulation) were tested for a complete pathway and falsified for a major pathway in an important model organism. We propose a subtler mechanism of flux regulation, with different roles for different enzymes, i.e., ''leader,'' ''follower,'' or ''conservative,'' the latter attempting to hold back the change in flux. This study makes this subtlety, so typical for biological systems, tractable experimentally and invites reformulation of the questions concerning the drives and constraints governing metabolic flux regulation.gene expression and metabolic regulation ͉ glycolysis ͉ regulation analysis ͉ metabolic control analysis T he flux through a metabolic pathway is determined by the activities of its enzymes and by their interactions with other enzymes. Metabolic-flux changes have often been observed in response to environmental or genetic changes. In the yeast Saccharomyces cerevisiae, for example, changes in glycolytic flux have frequently been found to be accompanied by a myriad of changes in glycolytic enzyme activities (e.g., 1, 2, this work) or amounts (3), which varied in magnitude and direction. The complexity of interactions between enzymes translates into a vast possibility space of combinations of enzyme-activity modulations leading to the same flux change. We wondered how the cell actually regulates its fluxes.Among the proposed mechanisms for metabolic-flux changes, the two clearest hypotheses are (i) modulation of single ratelimiting enzymes and (ii) multisite modulation, i.e., simultaneous and proportional modulation of all enzymes in the pathway, thus causing a change in flux while leaving metabolite concentrations unchanged (4). Although single rate-limiting enzymes exist, control of flux is quite often distributed over several enzymes (5). In the latter case, modulation of a single enzyme is likely to be an ineffective mechanism for changing a pathway's flux. Indeed, attempts to correlate flux changes with changes in single enzyme ac...
Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.The flux through a metabolic pathway depends on the kinetic characteristics and concentrations of the constituent enzymes, on the levels of co-enzymes, and on their compartmentation. The concentrations of the enzymes in turn depend on the rates of transcription, processing, nuclear export, translation, degradation of the mRNA, and protein processing and degradation. Because each of these levels can in principle be regulated, the challenge is how to analyze the behavior of such a complex system in terms of the underlying processes.Metabolic control analysis (MCA) 4 and extensions that include gene expression is a powerful approach for the analysis of complex biochemical networks (1-4). In MCA, the control exerted by an enzyme on a concentration of any substance X (5) is quantified by its concentration control coefficient, which is defined as the percentage increase of the steady-state concentration of X that results from a 1% activation of the enzyme of interest. The sum of the concentration control coefficients of all the enzymes in the network is 0. This reflects that (i) activation of some enzymes increases the concentration of X, whereas activation of other enzymes should reduce the concentration of X (these enzymes have negative concentration control coefficients), and (ii) the positive controls together are equal to the negative controls. The principles of MCA do not only apply to metabolic pathways; they can also be used and extended to dissect the control distribution in regulatory pathways beyond steady state (for example see Refs.6 and 7) or gene expression cascades (for example see e.g. Ref. 8). Earlier MCA, as also applied to trypanosomes, has looked more at the control of fluxes, showing that usually flux control coefficients are smaller than 1, because several enzymes partially...
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