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.Trypanosomatid parasites (Trypanosoma and Leishmania) are responsible for serious diseases of mankind in tropical and subtropical countries worldwide. These diseases affect millions of people, and hundreds of millions are at risk of becoming infected. Unfortunately, available treatments are largely inadequate. Currently used drugs are inefficacious and toxic. There is a desperate need for new effective and safe drugs, particularly in view of the development and spreading of drug resistance (1).Glycolysis plays an important role in the energy metabolism of these protozoan organisms, notably of Trypanosoma brucei when it lives in the blood of its mammalian host, causing a disease called sleeping sickness or human African trypanosomiasis in man and nagana in cattle (2, 3). This bloodstream form of T. brucei is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply. Oxidative metabolism involving mitochondrial Krebs cycle enzymes and oxidative phosphorylation are largely repressed. Therefore, glycolysis has been perceived as a potentially good target for anti-trypanosome drugs. Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner: the majority of the glycolytic enzymes are sequester...