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 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 P(i), several metabolites were identified as important new effectors. PP(i), 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 degrees C, 10 min), 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.
Villadsen
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Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark.
Nielsen
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TH
D017360:Arabidopsis / Q000201:enzymology*; D002850:Chromatography, Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005635:Fructosediphosphates / Q000378:metabolism*; D006358:Hot Temperature; D025481:Phosphofructokinase-2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D005635:Fructosediphosphates; C027652:fructose 2,6-diphosphate; D025481:Phosphofructokinase-2