Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Abstract

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...

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

“…IC 50 s for ATP and PP i in the acetate-forming direction were determined to be 0.81 Ϯ 0.17 mM for ATP and 0.83 Ϯ 0.27 mM for PP i . The estimated intracellular concentrations of ATP and PP i in E. histolytica trophozoites grown in the presence of glucose are 5 Ϯ 2 mM and 0.45 mM, respectively (35), suggesting that regulation of the acetate-forming activity of ACD by ATP and PP i is biologically relevant. The mode of inhibition by each of these compounds was determined by kinetic analysis in which the concentration of inhibitor was varied versus one substrate and the concentrations of the other two substrates were held constant.…”

“…Reported activities are means Ϯ SD of three replicates. GDP (3.3 Ϯ 1.2 mM versus 0.7 mM, respectively [35]). Furthermore, the intracellular concentration for ADP is above the K m value for ADP, whereas the intracellular GDP concentration is below the K m value.…”

“…The fact that the intracellular concentrations of ATP and GTP (5 Ϯ 2 mM and 1.8 mM, respectively [35]) are both below the K m values for these substrates suggests that the acetate/ATP-forming direction of the reaction is favored in the cell, even though the purified enzyme has similar activity in both directions. The higher catalytic efficiency observed with either substrate in the acetate-forming direction versus the acetyl-CoA-forming direction is consistent with this.…”

“…Although metabolic flux analysis using kinetic modeling suggested that ACD has little regulatory effect over glycolysis (35), its roles in amino acid degradation were not taken into account. On the contrary, one might expect that ACD's position in the extended glycolytic pathway of E. histolytica makes it a prime site for regulation in order to properly maintain ATP and acetyl-CoA concentrations.…”

Discovery of Entamoeba histolytica hexokinase 1 inhibitors through homology modeling and virtual screening

“…Another difference is hexokinase (HK) which is not inhibited by its product glucose-6-phosphate (G6P) like some vertebrate HKs 14 , instead, it is inhibited by physiological concentrations of AMP and ADP 13,15 . Furthermore, metabolic flux studies showed that HK controls 73% of the flux in E. histolytica glycolysis 16 . Therefore, EhHK is an excellent target in the search of specific inhibitors that can be developed into new drugs for the treatment of amebiasis.…”