*2.12. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Activity*

The reduction of NAD<sup>+</sup> to NADH was used to assess the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglicerate, as described before [44], with slight modifications. Total epimastigote lysates were incubated for 15 min at 37 ◦C in reaction media containing 100 mM Triethalonamine:HCl buffer (pH 7.5) containing 1.0 mM EDTA, 5 mM MgSO4, 1.0 mM dithiothreitol, 1.5 mM NAD+, and 30 mM KH2AsO4 [45,46]. The reaction was started using 2 mM glyceraldehyde-3-phosphate, and the absorbance at 340 nm was measured every 1 min for 5 min. Total NADH generation was determined using the NADH standard curve.

#### *2.13. Glucokinase Activity*

Cellular extracts of epimastigotes were incubated in a reaction buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM glucose, 1 unit/mL glucose-6-phosphate dehydrogenase (G6PDH) (*Leuconostoc mesenteroides*), 0.1% Triton X-100, 1 mM NaF, 5 mM NaN3, 1 mM ATP, and 50–100 μg/mL protein [47]. After 3 min of incubation, the reactions were started by the addition of 0.5 mM β-NADP+, and quantified spectrophotometrically following the reduction of β-NADP<sup>+</sup> to β-NADPH (λ = 340 nm) for 10 min. Total NADPH generation was determined using the NADPH standard curve.
