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.

To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F 0 -F 1 -ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31 P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.Prior work considered that mitochondria behave as a transducer with approximately first order response characteristics (1-4). This means that the response of mitochondrial oxidative phosphorylation (MOP) 1 to a stimulus would follow an approximately hyperbolic relation according to a Michaelis-Menten mechanism for the signal transduction (2, 3). With this understanding, the hypothesis that mitochondria detect variations in ATP utilization simply by sensing the variation in cytosolic [ADP] (2, 3) had to be discarded as a general mechanism after studies of the in situ dog heart showed 2-fold increases in MOP flux without much change in [ADP] (4). These observations led to consideration of alternative signals but not alternative kinetic functions of ADP-mediated signal transduction (1, 4). This was unfortunate, because earlier work on isolated mitochondria had shown that the response of MOP to changes in [ADP] is not hyperbolic (5, 6). Therefore, it remains possible that a higher order kinetic function for extramitochondrial [ADP] stimulation of MOP is responsible for the maintenance of energy balance in the mammalian cell.Here, we studied cytosolic [ADP] transduction in an intact cellular system. We used a general and unbiased analysis to test the apparent kinetic order of the transduction function. The generality of the in vivo result is tested against published kinetics of ADP stimulation of MOP in various other systems, and its implications for understanding the biochemistry of mitochondria and the integrative physiology of mitochondrial function in the cell are discussed. MATERIALS AND METHODS 31P NMR Spectroscopy of Intact Muscle-Phosphocreatine (PCr), P i , and ATP 31 P NMR resonances in well perfused human forearm flexor muscle of six consenting, healthy adult volunteers (five males and one female; age, 28 -55 years) were measured using high time resolution (7 s) 31 P NMR spectroscopy, and data acquisition and analysis methods developed in this laboratory (7, 8). 31P NMR spectra were collected using a CSI spectrometer operating at 2 tesla (General Electric). Different energy balance states were imposed by supramaximal percutaneous nerve stimu...

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Increasing the expression of various glycolytic operons in Zymomonss mobilis caused a significant decrease rather than increase in the glycolytic flux and growth rate. Because the relative decrease depended on the amount of overexpressed protein, and was independent of which enzyme was overexpressed, we attributed it to a protein burden effect. More specifically, we examined if the decrease in glycolytic flux could be explained by a decreased concentration of other glycolytic enzymes (for which glucokinase was used as a marker enzyme). Using the summation theorem of metabolic control theory we predicted the extent of this protein burden effect. The predictions were in good agreement with the experimental observations. This suggests that the negative flux control is caused either by a simple competition of the overexpressed gene with the expression of all other genes or by simple dilution. Furthermore, we determined the implications of protein burden for the determination of the extent to which an enzyme limits a flux. We conclude that a protein burden can cause a significant underestimation of the flux control coefficient, especially if the enzyme under investigation is a highly expressed enzyme.Keywords : protein burden, Zymomonas mobilis, flux control, glycolysis, metabolic control analysis INTRODUCTIONAttempts to optimize metabolic processes, by overexpression of enzymes that are thought to be important in the determination of the overall rate of formation of the desired product, often lead to negative results (Schaaff e t al., 1989;Niederberger et a/., 1992). This can be due to:(1) an intuitive overestimation of the actual importance of the enzyme in the production process -a clear distinction should be made between essential and controlling enzymes (Jensen e t a/., 1993a, b); (2) a shift of control upon overexpression (De Hollander, 1994;Small & Kacser, 1993); or (3) additional effects caused by the overexpression (Bailey, 1993). In the first case a quan- titative metabolic control analysis should lead to the identification of the enzymes that do exert flux control (Kacser & Burns, 1973;Heinrich & Rapoport, 1974; Groen e t a/., 1982; Fell, 1992). In the second case combined overexpression of a group (module, Schuster e t al., 1993) of enzymes should help (Small & Kacser, 1993). In the third case, the unspecific negative effects can be divided into energetic effects (costs to produce extra protein) and competitive effects (if the proteinsynthesizing machinery is limiting). In this paper we will use the term protein burden for the negative effect on any part of cell function caused by the overexpression of a protein independent of its catalytic activity. In order to evaluate the importance of an enzyme for the control of any flux under study, it is important to distinguish specific negative effects of the catalytic activity from this 'nonspecific ' protein burden. Thus, a quantitative analysis of the negative effects of expression of recombinant protein is needed. The protein burden effect has been recognize...