/* * Experimental and in Silico Analyses of Glycolytic Flux Control * in Bloodstream Form Trypanosoma brucei * * Model Status * * This CellML model runs in PCenv, COR and OpenCell and the units * are consistent throughout. It is unclear at this stage at to * whether it recreates the published results. It was adapted from * the Biomodels Database with the help of Lukas Endler (available: * http://www.ebi.ac.uk/biomodels-main/BIOMD0000000211). * * Model Structure * * ABSTRACT: A mathematical model of glycolysis in bloodstream * form Trypanosoma brucei was developed previously on the basis * of all available enzyme kinetic data (Bakker, B. M., Michels, * P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. * Biol. Chem. 272, 3207-3215). The model predicted correctly the * fluxes and cellular metabolite concentrations as measured in * non-growing trypanosomes and the major contribution to the flux * control exerted by the plasma membrane glucose transporter. * Surprisingly, a large overcapacity was predicted for hexokinase * (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). * Here, we present our further analysis of the control of glycolytic * flux in bloodstream form T. brucei. First, the model was optimized * and extended with recent information about the kinetics of enzymes * and their activities as measured in lysates of in vitro cultured * growing trypanosomes. Second, the concentrations of five glycolytic * enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) * in trypanosomes were changed by RNA interference. The effects * of the knockdown of these enzymes on the growth, activities, * and levels of various enzymes and glycolytic flux were studied * and compared with model predictions. Data thus obtained support * the conclusion from the in silico analysis that HXK, PFK, and * PYK are in excess, albeit less than predicted. Interestingly, * depletion of PFK and enolase had an effect on the activity (but * not, or to a lesser extent, expression) of some other glycolytic * enzymes. Enzymes located both in the glycosomes (the peroxisome-like * organelles harboring the first seven enzymes of the glycolytic * pathway of trypanosomes) and in the cytosol were affected. These * data suggest the existence of novel regulatory mechanisms operating * in trypanosome glycolysis. * * The original paper reference is cited below: * * Experimental and in silico analyses of glycolytic flux control * in bloodstream form Trypanosoma brucei, Marie-Astrid Albert, * Jurgen R. Haanstra, Veronique Hannaert, Joris Van Roy, Fred * R. Opperdoes, Barbara M. Bakker, and Paul A. M. Michels, 2005, * Journal of Biological Chemistry, volume 280, 28306-28315. PubMed * ID: 15955817 */ import nsrunit; unit conversion on; unit minute=60 second^1; unit mM=1 meter^(-3)*mole^1; unit mM_per_minute=.01666667 meter^(-3)*second^(-1)*mole^1; unit rate=1.6666667E-5 kilogram^(-1)*second^(-1)*mole^1; unit ul_per_mg=.001 kilogram^(-1)*meter^3; unit per_mM_per_minute=.01666667 meter^3*second^(-1)*mole^(-1); unit nanomole_per_mM2_per_minute_per_mg=1.6666667E-5 kilogram^(-1)*meter^6*second^(-1)*mole^(-1); unit ul_per_mg_per_minute=1.6666667E-5 kilogram^(-1)*meter^3*second^(-1); unit ul_per_mg_per_mM_per_minute=1.6666667E-5 kilogram^(-1)*meter^6*second^(-1)*mole^(-1); math main { realDomain time minute; time.min=0; extern time.max; extern time.delta; real total_volume ul_per_mg; total_volume=5.7; real glycosomal_volume ul_per_mg; real cytosolic_volume ul_per_mg; real pyruvate_cyt(time) mM; when(time=time.min) pyruvate_cyt=10; real adp_cyt(time) mM; when(time=time.min) adp_cyt=1.31652277625; real atp_cyt(time) mM; when(time=time.min) atp_cyt=0.341738611875; real phosphoenolpyruvate_cyt(time) mM; when(time=time.min) phosphoenolpyruvate_cyt=0; real twophosphoglycerate_cyt(time) mM; when(time=time.min) twophosphoglycerate_cyt=0; real amp_cyt(time) mM; when(time=time.min) amp_cyt=2.24173861188; real threephosphoglycerate_cyt(time) mM; when(time=time.min) threephosphoglycerate_cyt=0; real dihydroxyacetonephosphate_cyt(time) mM; when(time=time.min) dihydroxyacetonephosphate_cyt=2.23134594788; real glycerol3phosphate_cyt(time) mM; when(time=time.min) glycerol3phosphate_cyt=2.76865405212; real v_PT(time) rate; real v_PK(time) rate; real v_AKc(time) rate; real v_AU(time) rate; real v_ENO(time) rate; real v_PGM(time) rate; real v_PGT(time) rate; real v_ANTI(time) rate; real v_GPO(time) rate; real glucose_gly(time) mM; when(time=time.min) glucose_gly=0; real atp_gly(time) mM; when(time=time.min) atp_gly=0.240501857508; real adp_gly(time) mM; when(time=time.min) adp_gly=1.51899628498; real amp_gly(time) mM; when(time=time.min) amp_gly=4.24050185751; real glucose6phosphate_gly(time) mM; when(time=time.min) glucose6phosphate_gly=0.5; real fructose6phosphate_gly(time) mM; when(time=time.min) fructose6phosphate_gly=0.5; real fructose16bisphosphate_gly(time) mM; when(time=time.min) fructose16bisphosphate_gly=10; real dihydroxyacetonephosphate_gly(time) mM; when(time=time.min) dihydroxyacetonephosphate_gly=8.47911460193; real glyceraldehyde3phosphate_gly(time) mM; when(time=time.min) glyceraldehyde3phosphate_gly=2.5; real nad_gly(time) mM; when(time=time.min) nad_gly=2; real nadh_gly(time) mM; when(time=time.min) nadh_gly=2; real bisphosphoglycerate_gly(time) mM; when(time=time.min) bisphosphoglycerate_gly=0.5; real glycerol3phosphate_gly(time) mM; when(time=time.min) glycerol3phosphate_gly=10.5208853981; real threephosphoglycerate_gly(time) mM; when(time=time.min) threephosphoglycerate_gly=0; real glycerol_gly(time) mM; when(time=time.min) glycerol_gly=0; real v_GT(time) rate; real v_HK(time) rate; real v_AKg(time) rate; real v_GK(time) rate; real v_PFK(time) rate; real v_PGK(time) rate; real v_PGI(time) rate; real v_ALD(time) rate; real v_GPDH(time) rate; real v_GAPDH(time) rate; real v_TPI(time) rate; real v_GlyT(time) rate; real glucose_external(time) mM; when(time=time.min) glucose_external=5; real pyruvate_ext(time) mM; when(time=time.min) pyruvate_ext=0; real glycerol_ext(time) mM; when(time=time.min) glycerol_ext=0; real Vmax_v1 rate; Vmax_v1=108.9; real KGlc mM; KGlc=1; real Alpha_v1 dimensionless; Alpha_v1=0.75; real Vmax_v2 rate; Vmax_v2=1929; real KGlcInt_v2 mM; KGlcInt_v2=0.1; real KATPg_v2 mM; KATPg_v2=0.116; real KADPg_v2 mM; KADPg_v2=0.126; real KGlc6P_v2 mM; KGlc6P_v2=12; real Kms_vPGI mM; Kms_vPGI=0.4; real Kmp_vPGI mM; Kmp_vPGI=0.12; real Vf_vPGI rate; Vf_vPGI=1305; real Vr_vPGI rate; Vr_vPGI=1305; real Vmax_v4 rate; Vmax_v4=1708; real Ki1Fru16BP_v4 mM; Ki1Fru16BP_v4=15.8; real KFru6P_v4 mM; KFru6P_v4=0.82; real KATPg_v4 mM; KATPg_v4=0.026; real Ki2Fru16BP_v4 mM; Ki2Fru16BP_v4=10.7; real Vmax_v5 rate; Vmax_v5=560; real Keq_v5 mM; Keq_v5=0.069; real r_v5 dimensionless; r_v5=1.19; real KGAP_v5 mM; KGAP_v5=0.067; real KGAPi_v5 mM; KGAPi_v5=0.098; real Kms_vTPI mM; Kms_vTPI=1.2; real Kmp_vTPI mM; Kmp_vTPI=0.25; real Vf_vTPI rate; Vf_vTPI=999.3; real Vr_vTPI rate; Vr_vTPI=5696.01; real Vmax_v7 rate; Vmax_v7=720.9; real KGAP_v7 mM; KGAP_v7=0.15; real KNAD_v7 mM; KNAD_v7=0.45; real r_v7 dimensionless; r_v7=0.67; real KBPGA13_v7 mM; KBPGA13_v7=0.1; real KNADH_v7 mM; KNADH_v7=0.02; real Vmax_v8 rate; Vmax_v8=465; real KDHAPg_v8 mM; KDHAPg_v8=0.1; real KNADH_v8 mM; KNADH_v8=0.01; real r_v8 dimensionless; r_v8=0.28; real KNAD_v8 mM; KNAD_v8=0.4; real KGly3Pg_v8 mM; KGly3Pg_v8=2; real Km_GPO mM; Km_GPO=1.7; real V_GPO_0 rate; V_GPO_0=368; real Km_PT mM; Km_PT=1.96; real V_PT_0 rate; V_PT_0=200; real Vmax_v11 rate; Vmax_v11=2862; real KBPGA13_v11 mM; KBPGA13_v11=0.003; real KADPg_v11 mM; KADPg_v11=0.1; real r_v11 dimensionless; r_v11=0.47; real KPGA3_v11 mM; KPGA3_v11=1.62; real KATPg_v11 mM; KATPg_v11=0.29; real Vmax_v12 rate; Vmax_v12=1020; real PK_n dimensionless; PK_n=2.5; real KADP_v12 mM; KADP_v12=0.114; real k_vAU rate; k_vAU=50; real Vmax_v14 rate; Vmax_v14=200; real KGly3Pg_v14 mM; KGly3Pg_v14=3.83; real KADPg_v14 mM; KADPg_v14=0.56; real r_v14 dimensionless; r_v14=60.86; real KGlycerol_v14 mM; KGlycerol_v14=0.44; real KATPg_v14 mM; KATPg_v14=0.24; real Kms_vPGM mM; Kms_vPGM=0.27; real Kmp_vPGM mM; Kmp_vPGM=0.11; real Vf_vPGM rate; Vf_vPGM=225; real Vr_vPGM rate; Vr_vPGM=495; real Kms_vENO mM; Kms_vENO=0.054; real Kmp_vENO mM; Kmp_vENO=0.24; real Vf_vENO rate; Vf_vENO=598; real Vr_vENO rate; Vr_vENO=394.68; real k_vAKc nanomole_per_mM2_per_minute_per_mg; k_vAKc=1000000; real keqakc dimensionless; keqakc=0.442; real k_vAKg nanomole_per_mM2_per_minute_per_mg; k_vAKg=1000000; real keqakg dimensionless; keqakg=0.442; real k1_vPGT ul_per_mg_per_minute; k1_vPGT=1000000; real k2_vPGT ul_per_mg_per_minute; k2_vPGT=1000000; real v_ANTI.k1 ul_per_mg_per_mM_per_minute; v_ANTI.k1=1000000; real v_ANTI.k2 ul_per_mg_per_mM_per_minute; v_ANTI.k2=1000000; real v_GlyT.k1 ul_per_mg_per_minute; v_GlyT.k1=1000000; real v_GlyT.k2 ul_per_mg_per_minute; v_GlyT.k2=1000000; // glycosomal_volume=(total_volume*.043); cytosolic_volume=(total_volume*.957); // pyruvate_cyt:time=((-1)*1*v_PT/cytosolic_volume+1*v_PK/cytosolic_volume); adp_cyt:time=((-1)*1*v_PK/cytosolic_volume+1*v_AU/cytosolic_volume+2*v_AKc/cytosolic_volume); atp_cyt:time=(1*v_PK/cytosolic_volume-1*v_AU/cytosolic_volume-1*v_AKc/cytosolic_volume); phosphoenolpyruvate_cyt:time=((-1)*1*v_PK/cytosolic_volume+1*v_ENO/cytosolic_volume); twophosphoglycerate_cyt:time=(1*v_PGM/cytosolic_volume-1*v_ENO/cytosolic_volume); amp_cyt:time=((-1)*1*v_AKc/cytosolic_volume); threephosphoglycerate_cyt:time=((-1)*1*v_PGM/cytosolic_volume+1*v_PGT/cytosolic_volume); dihydroxyacetonephosphate_cyt:time=((-1)*1*v_ANTI/cytosolic_volume+1*v_GPO/cytosolic_volume); glycerol3phosphate_cyt:time=(1*v_ANTI/cytosolic_volume-1*v_GPO/cytosolic_volume); // glucose_gly:time=(1*v_GT/glycosomal_volume-1*v_HK/glycosomal_volume); atp_gly:time=((-1)*1*v_HK/glycosomal_volume-1*v_PFK/glycosomal_volume+1*v_PGK/glycosomal_volume+1*v_GK/glycosomal_volume-1*v_AKg/glycosomal_volume); adp_gly:time=(1*v_HK/glycosomal_volume+1*v_PFK/glycosomal_volume-1*v_PGK/glycosomal_volume-1*v_GK/glycosomal_volume+2*v_AKg/glycosomal_volume); amp_gly:time=((-1)*1*v_AKg/glycosomal_volume); glucose6phosphate_gly:time=(1*v_HK/glycosomal_volume-1*v_PGI/glycosomal_volume); fructose6phosphate_gly:time=(1*v_PGI/glycosomal_volume-1*v_PFK/glycosomal_volume); fructose16bisphosphate_gly:time=(1*v_PFK/glycosomal_volume-1*v_ALD/glycosomal_volume); dihydroxyacetonephosphate_gly:time=(1*v_ALD/glycosomal_volume-1*v_TPI/glycosomal_volume-1*v_GPDH/glycosomal_volume+1*v_ANTI/glycosomal_volume); glyceraldehyde3phosphate_gly:time=(1*v_ALD/glycosomal_volume-1*v_GAPDH/glycosomal_volume+1*v_TPI/glycosomal_volume); nad_gly:time=((-1)*1*v_GAPDH/glycosomal_volume+1*v_GPDH/glycosomal_volume); nadh_gly:time=(1*v_GAPDH/glycosomal_volume-1*v_GPDH/glycosomal_volume); bisphosphoglycerate_gly:time=(1*v_GAPDH/glycosomal_volume-1*v_PGK/glycosomal_volume); glycerol3phosphate_gly:time=(1*v_GPDH/glycosomal_volume-1*v_GK/glycosomal_volume-1*v_ANTI/glycosomal_volume); threephosphoglycerate_gly:time=(1*v_PGK/glycosomal_volume-1*v_PGT/glycosomal_volume); glycerol_gly:time=(1*v_GK/glycosomal_volume-1*v_GlyT/glycosomal_volume); // glycerol_ext:time=(0 mM_per_minute); glucose_external:time=(0 mM_per_minute); pyruvate_ext:time=(0 mM_per_minute); // v_GT=(Vmax_v1*((glucose_external-glucose_gly)/(KGlc+glucose_external+glucose_gly+Alpha_v1*glucose_external*glucose_gly/KGlc))); // v_HK=(Vmax_v2*(glucose_gly/KGlcInt_v2*(atp_gly/KATPg_v2)/((1+atp_gly/KATPg_v2+adp_gly/KADPg_v2)*(1+glucose_gly/KGlcInt_v2+glucose6phosphate_gly/KGlc6P_v2)))); // v_PGI=((Vf_vPGI*glucose6phosphate_gly/Kms_vPGI-Vr_vPGI*fructose6phosphate_gly/Kmp_vPGI)/(1+glucose6phosphate_gly/Kms_vPGI+fructose6phosphate_gly/Kmp_vPGI)); // v_PFK=(Vmax_v4*(Ki1Fru16BP_v4/(Ki1Fru16BP_v4+fructose16bisphosphate_gly))*(fructose6phosphate_gly/KFru6P_v4*(atp_gly/KATPg_v4)/((1+fructose6phosphate_gly/KFru6P_v4+fructose16bisphosphate_gly/Ki2Fru16BP_v4)*(1+atp_gly/KATPg_v4)))); // v_ALD=(Vmax_v5*(fructose16bisphosphate_gly-glyceraldehyde3phosphate_gly*dihydroxyacetonephosphate_gly/Keq_v5)/((.009 mM)*(1+atp_gly/(.68 mM)+adp_gly/(1.51 mM)+amp_gly/(3.65 mM))+fructose16bisphosphate_gly+glyceraldehyde3phosphate_gly*(.015 mM)*(1+atp_gly/(.68 mM)+adp_gly/(1.51 mM)+amp_gly/(3.65 mM))/Keq_v5*(1/r_v5)+dihydroxyacetonephosphate_gly*KGAP_v5/Keq_v5*(1/r_v5)+fructose16bisphosphate_gly*glyceraldehyde3phosphate_gly/KGAPi_v5+glyceraldehyde3phosphate_gly*dihydroxyacetonephosphate_gly/Keq_v5*(1/r_v5))); // v_TPI=((Vf_vTPI*dihydroxyacetonephosphate_gly/Kms_vTPI-Vr_vTPI*glyceraldehyde3phosphate_gly/Kmp_vTPI)/(1+dihydroxyacetonephosphate_gly/Kms_vTPI+glyceraldehyde3phosphate_gly/Kmp_vTPI)); // v_GAPDH=(Vmax_v7*(glyceraldehyde3phosphate_gly/KGAP_v7*(nad_gly/KNAD_v7)-r_v7*(bisphosphoglycerate_gly/KBPGA13_v7)*(nadh_gly/KNADH_v7))/((1+glyceraldehyde3phosphate_gly/KGAP_v7+bisphosphoglycerate_gly/KBPGA13_v7)*(1+nad_gly/KNAD_v7+nadh_gly/KNADH_v7))); // v_GPDH=(Vmax_v8*(dihydroxyacetonephosphate_gly/KDHAPg_v8*(nadh_gly/KNADH_v8)-r_v8*(nad_gly/KNAD_v8)*(glycerol3phosphate_gly/KGly3Pg_v8))/((1+dihydroxyacetonephosphate_gly/KDHAPg_v8+glycerol3phosphate_gly/KGly3Pg_v8)*(1+nadh_gly/KNADH_v8+nad_gly/KNAD_v8))); // v_GPO=(V_GPO_0*(glycerol3phosphate_cyt/Km_GPO/(1+glycerol3phosphate_cyt/Km_GPO))); // v_PT=(V_PT_0*pyruvate_cyt/(Km_PT+pyruvate_cyt)); // v_PGK=(Vmax_v11*(bisphosphoglycerate_gly/KBPGA13_v11*adp_gly/KADPg_v11-r_v11*threephosphoglycerate_gly/KPGA3_v11*atp_gly/KATPg_v11)/((1+bisphosphoglycerate_gly/KBPGA13_v11+threephosphoglycerate_gly/KPGA3_v11)*(1+adp_gly/KADPg_v11+atp_gly/KATPg_v11))); // v_PK=(Vmax_v12*(phosphoenolpyruvate_cyt/((.34 mM)*(1+atp_cyt/(.57 mM)+adp_cyt/(.64 mM))))^PK_n*adp_cyt/KADP_v12/((1+(phosphoenolpyruvate_cyt/((.34 mM)*(1+atp_cyt/(.57 mM)+adp_cyt/(.64 mM))))^PK_n)*(1+adp_cyt/KADP_v12))); // v_AU=(k_vAU*atp_cyt/adp_cyt); // v_GK=(Vmax_v14*(glycerol3phosphate_gly/KGly3Pg_v14*adp_gly/KADPg_v14-r_v14*glycerol_gly/KGlycerol_v14*atp_gly/KATPg_v14)/((1+glycerol3phosphate_gly/KGly3Pg_v14+glycerol_gly/KGlycerol_v14)*(1+adp_gly/KADPg_v14+atp_gly/KATPg_v14))); // v_PGM=((Vf_vPGM*threephosphoglycerate_cyt/Kms_vPGM-Vr_vPGM*twophosphoglycerate_cyt/Kmp_vPGM)/(1+threephosphoglycerate_cyt/Kms_vPGM+twophosphoglycerate_cyt/Kmp_vPGM)); // v_ENO=((Vf_vENO*twophosphoglycerate_cyt/Kms_vENO-Vr_vENO*phosphoenolpyruvate_cyt/Kmp_vENO)/(1+twophosphoglycerate_cyt/Kms_vENO+phosphoenolpyruvate_cyt/Kmp_vENO)); // v_AKc=(k_vAKc*(atp_cyt*amp_cyt-keqakc*adp_cyt*adp_cyt)); // v_AKg=(k_vAKg*(atp_gly*amp_gly-keqakg*adp_gly*adp_gly)); // v_PGT=(k1_vPGT*threephosphoglycerate_gly-k2_vPGT*threephosphoglycerate_cyt); // v_ANTI=(v_ANTI.k1*glycerol3phosphate_gly*dihydroxyacetonephosphate_cyt-v_ANTI.k2*glycerol3phosphate_cyt*dihydroxyacetonephosphate_gly); // v_GlyT=(v_GlyT.k1*glycerol_gly-v_GlyT.k2*glycerol_ext); }