Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. I. Hexokinase and phosphofructokinase.

Hexokinase (ATP: hexose 6-phosphotransferase, E.C.2.7.1.1) and phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11), two key regulatory enzymes of the glycolytic pathway in vertebrate cells, have been isolated and partially purified from Trypanosoma (Schizotrypanum) cruzi epimastigotes. Both enzymes are associated with particles sedimentable at 105 000 X gav for 1 h and have a high degree of latency; they can be solubilized by sonication. Hexokinase catalyses the phosphorylation of a series of monosaccharides at the following relative rates: D-glucose (100) congruent to D-fructose (97) greater than 2-deoxy-D-glucose (72) congruent to mannose (69) greater than 2-amino-D-glucose (63) greater than 3-O-methyl-D-glucose (21). Very little or no phosphorylating activity was found for D-galactose, N-acetyl-2-amino-D-glucose or 1-alpha-methyl-D-glucose. D-Glucose phosphorylation at fixed ATP concentration follows simple Michaelis-Menten kinetics with Km = 40 microM and Vmax = 440 nmol min-1 mg-1 protein. D-Mannose, 2-deoxy-D-glucose and N-acetyl-2-amino-D-glucose act as competitive inhibitors of glucose phosphorylation, suggesting a single kinase. Mg2+-ATP is the preferred phosphoryl donor, ITP and GTP being much less effective. T. cruzi hexokinase is not inhibited by D-glucose 6-phosphate, or by any of the following compounds (2 mM):D-fructose 6-phosphate, D-fructose 1,6-diphosphate, D-glucose 1,6-diphosphate, phosphoenol pyruvate, L-malate and citrate. Phosphofructokinase displays simple Michaelis-Menten kinetics with no evidence of sigmoidicity with respect to D-fructose 6-phosphate at all ATP concentrations tested, giving a Km of 1.31 mM and Vmax = 400 nmol min-1 mg-1 protein at optimal ATP levels. With respect to ATP, the enzyme exhibits Michaelis-Menten kinetics at low concentration (less than 1 mM) of the substrate (Km = 40 microM at 5 mM MgCl2, pH 7.4). A moderate inhibition is observed at high ATP levels (70% of maximal activity at 2 mM). GTP can substitute for ATP as the phosphoryl donor (Km = 79 microM under the same conditions), but produces only very small inhibitory effects at high concentrations. 5'-AMP activates the enzyme by decreasing its Km with respect to D-fructose 6-phosphate without affecting Vm. Other well-known regulators of the activity of this enzyme in procaryote and vertebrate systems such as citrate, phosphoenol pyruvate, ammonium and phosphate ions have no effect in T. cruzi.