Purification and physical properties of glucokinase from Thiobacillus versutus (A2)

Abstract

Glucokinase was purified 749-fold from Thiobacillus versutus. Polyacrylamide gel electrophoresis revealed the presence of five protein bands in the purified preparation. The purified enzyme retained its original activity after 4 weeks of storage at −20 °C, but not at higher temperatures. Glucose provided some protection at 25 °C. The optimum temperature for activity was between 20 and 25 °C, and an energy of activation (Ea) of 1.305 kcal/mol was calculated. A Q10 value of about 1.080 was determined over two 10 °C temperature ranges between 5 and 20 °C. With both Hepes and Tricine buffers, the optimum pH was 7.8. The enzyme was specific for glucose, with a Km of 0.86 mM. The most efficient phosphoryl donor was ATP with a Km of 0.78 mM. About 30% of the activity observed with ATP was obtained with equimolar amounts of ITP, while TTP and UTP gave 7.0 and 12%, respectively. The enzyme displayed an absolute requirement for a divalent cation, with Mg2+ (Km, 0.27 mM) being the most effective.