N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

Abstract

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase(F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and Pi, several metabolites were identified as important new effectors. PPi, phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42°C, 10min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.