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

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase) /fructose-2,6-bisphosphatase (F26BPase) catalyses the formation and degrada tion of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encodin g the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric proper ties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and P-i, several metabolites were identified as important new e ffectors. PP, 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 wer e rapidly inactivated by mild heat treatment (42 degreesC, 10 min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 34 5-amino-acid N-terminus of unknown function. The role of this region was ex amined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromato graphy. The full-length enzyme was eluted as a homotetramer, whereas the tr uncated enzymes were eluted as monomers. Deletion of the N-terminus decreas ed the kinase/phosphatase activity ratio by 4-fold, and decreased the affin ity for the substrate fructose 6-phosphate. The data show that the N-termin us is important both for subunit assembly and for defining the kinetic prop erties of the enzyme.