br Acknowledgements and disclosures This work was supported

Acknowledgements and disclosures This work was supported in part by the National Institutes of Health (R01 HL-131673-01A1) and the Veterans Administration (BX-002539-01), United States. The authors have nothing to disclose concerning any conflict of interest.
Introduction Adaptations are often evident in Auranofin receptor that are upstream in a metabolic pathway, or that are in the branching points of metabolic pathways (Wright and Rausher, 2010, Olson-Manning et al., 2013). Hexokinase is a prime example of how both conditions can be met. It is the glucose-phosphorylating enzyme, thus it initiates both the glycolysis pathway, leading to ATP production (main function), and the pentose phosphate pathway, leading to NADPH production; it is also the mannose-phosphorylating enzyme, thus it initiates the sugar nucleotide biosynthesis pathway, leading to glycoprotein production (Alton et al., 1998, Cárdenas et al., 1998, Sharma et al., 2014). African trypanosomes of the subgenus Trypanozoon include five species and subspecies that infect mammals. T. brucei brucei, occurring throughout Sub-Saharan Africa, and T. b. rhodesiense, occurring in eastern and southern Africa, cause nagana, mainly in ruminants. In addition, T. b. rhodesiense causes acute sleeping sickness in humans (Katunguka-Rwakishaya, 1996, Moloo et al., 1999, Van den Bossche et al., 2005, Anderson et al., 2011, Majekodunmi et al., 2013, Franco et al., 2014). T. b. gambiense, occurring in western and central Africa, causes chronic sleeping sickness in humans: it is responsible for 98% of the cases of human tripanosomiasis in Sub-Saharan Africa; wild mammals are minor reservoirs (Franco et al., 2014). T. evansi and T. equiperdum, which are widespread across the tropics and subtropics of the world, respectively cause surra and dourine in domestic and wild mammals, but some cases of human infection are known (Zablotskij et al., 2003, Desquesnes et al., 2013). The three subspecies of T. brucei are termed pleomorphic because they exist as “slender” (asexually multiplicative) and “stumpy” (non-multiplicative) forms in mammalian blood, and as a procyclic (sexual) form in the endemic Sub-Saharan tsetse flies, which are their vectors (Franco et al., 2014). T. evansi and T. equiperdum are termed monomorphic because they exist only as the “slender” form in mammalian blood. T. evansi is transmitted mechanically in blood micro-volumes carried in the mouthparts of several genera of hematophagous flies, whereas T. equiperdum is transmitted as a venereal disease without the participation of insect vectors (Zablotskij et al., 2003, Desquesnes et al., 2013). Lack of dependence on tsetse flies may have been a crucial trait for trypanosomes of the subgenus Trypanozoon to spread out of their native Sub-Saharan Africa (Lun and Desser, 1995). To obtain energy from host blood glucose, “slender” trypanosomes of the subgenus Trypanozoon are fully dependent on their glycolytic capability (Marshall, 1948, Ryley, 1956, Grant and Fulton, 1957, Bringaud et al., 2006, Moreno et al., 2015, Moreno and Nava, 2015). This fact has caused trypanosomal glycolytic enzymes to be the subject of much attention as potential targets for chemotherapeutic drugs (Bakker et al., 1997, Bakker et al., 1999a, Bakker et al., 1999b, Kotsikorou et al., 2006, Chambers et al., 2008a). Knowledge of the variability in the kinetic properties of hexokinases within and among members of Trypanozoon is limited. In particular, nothing is known on this topic for the monomorphic members of the subgenus. This is surprising given that hexokinase is the most upstream enzyme in three major metabolic pathways, and given that the kinetic properties of enzymes are fundamental to understand the rate and specificity of metabolic processes. Hexokinase is assumed to be similar in molecular weight in the bloodstream form of all members of Trypanozoon (Risby and Seed, 1969). In T. brucei, hexokinase is a hexamer, with a native molecular weight of 295 KDa, and a subunit molecular weight of 50.3 KDa (Misset et al., 1986). In T. brucei (both bloodstream and procyclic), hexokinase is composed of two forms, hexokinase 1 (HK1) and hexokinase 2 (HK2), sharing 97.7% of their amino acid identity (Colasante et al., 2006). HK1 uses glucose, mannose, and fructose as substrates, and is not inhibited by inorganic pyrophosphate (PPi). HK2 lacks activity, but its presence causes the inhibition of HK1 by PPi (Morris et al., 2006, Chambers et al., 2008b). Although hexokinase lacks a classic regulation, it has been proposed that HK2 might have a regulatory role (Nwagwu and Opperdoes, 1982, Chambers et al., 2008b).