Characterization of the separate kinase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two distinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate domain or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized, The two forms of the domain with different lengths (amino acids 1 similar to 249 and 1 similar to 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a k(cat) 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain of the bifunctional enzyme is far less perfect in structure in the absence of the bisphosphatase domain, though it still possesses the 6-phosphofructo-2-kinase activity.