Kinetic mechanism and some properties of glucose-6-phosphate dehydrogenase from sheep brain cortex

Abstract

Objective: Glucose-6-phosphate dehydrogenase is an important key enzyme which catalyzes the first oxidative step of the pentose phosphate pathway. The objectives of this study are to determine some properties and kinetic mechanism of glucose-6-phosphate dehydrogenase. Method: In this study we used the enzyme that was purified from sheep brain cortex. The kinetic analysis and other studies were performed within three days after the purification to prevent any loss of enzyme activity. Results: The subunit molecular weight was determined as 61.2 kDa by SDS-PAGE and 64.5 kDa by Sephadex G-200 gel filtration chromatography. The activation energy (Ea) of the reaction was calculated from the Arrhenius plot as 6.689 kcal/mol. We have calculated the temperature coefficient as 1.913 from the integrated form of the Arrhenius equation (for the temperatures between 30 - 40 oC). Optimum pH of the enzyme was determined as 8. Kinetic constants estimated by a nonlinear curve-fitting program are as follows: Vm= 2.182 ± 0.064 mmol/min/mg enzyme; Km NADP+ = 0.064 ± 0.001 mM; Km G6P = 0.0729 ± 0.006 mM; Ki G6P = 0.074 ± 0.003 mM; Ki NADP+ = 0.056 ± 0.001mM. Conclusion: Sheep brain cortex glucose-6-phosphate dehydrogenase exhibits classical Michaelis-Menten kinetics. Product inhibition studies were undertaken to distinguish the type of the mechanism. We have found that G6PD enzyme obeys 'Theorell-Chance' mechanism. The kinetic behavior, molecular weight, optimum temperature and pH of the enzyme were in accordance with our previous studies.