Glucose 6-phosphate dehydrogenase from larval Taenia crassiceps (cysticerci): purification and properties

Abstract

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity from the soluble fraction of larval Taenia crassiceps (Eucestoda: Cyclophyllidea) by a three-step protocol. Specific activity of the pure enzyme was 33.8 ± 2.1 U mg⁻¹ at 25°C and pH 7.8 with d-glucose 6-phosphate and NADP⁺ as substrates. The activity increases to 67.6 ± 3.9 U mg⁻¹ at 39°C, a more physiological temperature in the intermediary host. Enzyme activity was maximal between pH 6.7 and 7.8. K _m values were 14 ± 1.7 μM and 1.3 ± 0.4 μM for glucose 6-phosphate and NADP⁺, respectively. The enzyme showed absolute specificity for its sugar substrate. NAD⁺ was also a substrate but with a low catalytic efficiency (207 M⁻¹ s⁻¹). No essential requirement for Mg⁺⁺ or Ca⁺⁺ was observed. Relative molecular mass of the native enzyme was 134,000 ± 17,200, while a value of 61,000 ± 1,700 was obtained for the enzyme subunit. Thus, glucose 6-phosphate dehydrogenase from T. crassiceps exists as a dimeric protein. The enzyme’s isoelectric point was 4.5. The enzyme’s activity dependence on temperature was complex, resulting in a biphasic Arrhenius plot. Activation energies of 9.91 ± 0.51 and 7.94 ± 0.45 kcal mol⁻¹ were obtained. Initial velocity patterns complemented with inhibition studies by product and substrate’s analogues support a random bi bi sequential mechanism in rapid equilibrium. The low K _i value of 1.95 μM found for NADPH suggests a potential regulatory role for this nucleotide.