Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei Export

@article{citeulike:2775971, 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. 10.1074/jbc.M502403200}, author = {Albert, Marie-Astrid and Haanstra, Jurgen R. and Hannaert, Veronique and Van Roy, Joris and Opperdoes, Fred R. and Bakker, Barbara M. and Michels, Paul A.}, citeulike-article-id = {2775971}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M502403200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/280/31/28306}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15955817}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15955817}, day = {5}, doi = {10.1074/jbc.M502403200}, journal = {J. Biol. Chem.}, keywords = {glycolysis, kinetic-model, trypanosome}, month = {August}, number = {31}, pages = {28306--28315}, posted-at = {2008-05-09 15:16:55}, priority = {2}, title = {Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei}, url = {http://dx.doi.org/10.1074/jbc.M502403200}, volume = {280}, year = {2005} }

TY - JOUR ID - citeulike:2775971 L3 - citeulike-article-id:2775971 N2 - 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. 10.1074/jbc.M502403200 IS - 31 JF - J. Biol. Chem. EP - 28315 TI - Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei VL - 280 SP - 28306 KW - glycolysis KW - kinetic-model KW - trypanosome AU - Albert, Marie-Astrid AU - Haanstra, Jurgen AU - Hannaert, Veronique AU - Van Roy, Joris AU - Opperdoes, Fred AU - Bakker, Barbara AU - Michels, Paul PY - 2005/08/05/ UR - http://dx.doi.org/10.1074/jbc.M502403200 ER -