Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.

Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphatedependent (over ATP-dependent) enzymes. To determine the flux-control distribution of glycolysis and understand its underlying control mechanisms, a kinetic model of the pathway was constructed by using the software gepasi. The model was based on the kinetic parameters determined in the purified recombinant enzymes, and the enzyme activities, and steady-state fluxes and metabolite concentrations determined in amoebal trophozoites. The model predicted, with a high degree of accuracy, the flux and metabolite concentrations found in trophozoites, but only when the pyrophosphate concentration was held constant; at variable pyrophosphate, the model was not able to completely account for the ATP production ⁄ consumption balance, indicating the importance of the pyrophosphate homeostasis for amoebal glycolysis. Control analysis by the model revealed that hexokinase exerted the highest flux control (73%), as a result of its low cellular activity and strong AMP inhibition. 3-Phosphoglycerate mutase also exhibited significant flux control (65%) whereas the other pathway enzymes showed little or no control. The control of the ATP concentration was also mainly exerted by ATP consuming processes and 3-phosphoglycerate mutase and hexokinase (in the producing block). The model also indicated that, in order to diminish the amoebal glycolytic flux by 50%, it was required to decrease hexokinase or 3-phosphoglycerate mutase by 24% and 55%, respectively, or by 18% for both enzymes. By contrast, to attain the same reduction in flux by inhibiting the pyrophosphate-dependent enzymes pyrophosphate-phosphofructokinase and pyruvate phosphate dikinase, they should be decreased > 70%. On the basis of metabolic control analysis, steps whose inhibition would have stronger negative effects on the energy metabolism of this parasite were identified, thus becoming alternative targets for drug design.Abbreviations ADH, alcohol dehydrogenase; AK, adenylate kinase; ALDO, fructose 1,6-bisphosphate aldolase; AldDH, aldehyde dehydrogenase; ATP-PFK, ATP-dependent phosphofructokinase; DHAP, dihydroxyacetone phosphate; ENO, enolase; EtOH, ethanol; F6P, fructose 6-phosphate; F(1,6)P 2 , fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Gly3PDH, glycerol 3-phosphate dehydrogenase; HK, hexokinase; HPI, hexose 6-phosphate isomerase; HXT, hexose transporter; LDH, lactate dehydrogenase; MCA, metabolic control analysis; PGAM, 3-phosphoglycerate mutase; PGK, phosphoglycerate kinase; PGM, phosphoglucomutase; 3PGDH, 3-phosphoglycerate dehydrogenase; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PPi, pyrophosphate; PPi-PFK, pyrophosphate-dependent phosphofructokinase; PPP, pentose phosphate pathway; PFOR, pyruvate:ferredoxin oxidoreductase; PFOR-AldDH, lumped reacti...

The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O 2 exposure (IC 50 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe 2+ . CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O 2 exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe 2+ -reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O 2 exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress. Abbreviations

The synthesis of ATP in the human parasite Entamoeba histolytica is carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose‐6‐phosphate isomerase, inorganic pyrophosphate‐dependent phosphofructokinase, fructose‐1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes from E. histolytica. Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. An in vitro reconstruction of the final stages of glycolysis was made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH.