Molecular and kinetic characterisation of sugarcane pyrophosphate: fructose-6-phosphate 1-phosphotransferase and its possible role in the sucrose accumulation phenotype
Jan-Hendrik Groenewald A C and Frederik Coenraad Botha A BA Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
B South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe 4300, South Africa.
C Corresponding author. Email: jhgr@sun.ac.za
D This paper originates from a presentation at the 8th International Congress of Plant Molecular Biology, Adelaide, Australia, August 2006.
Functional Plant Biology 34(6) 517-525 https://doi.org/10.1071/FP06213
Submitted: 1 September 2006 Accepted: 29 March 2007 Published: 1 June 2007
Abstract
The amount of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in sugarcane internodal tissue is inversely correlated with sucrose content. To help elucidate this apparent role of PFP in sucrose accumulation in sugarcane we have determined its molecular and kinetic properties. Sugarcane PFP was purified 285-fold to a final specific activity of 4.23 µmol min–1 mg–1 protein. It contained two polypeptides of 63.2 and 58.0 kDa respectively, at near equal amounts that cross-reacted with potato PFP-α and –β antiserum. In gel filtration analyses the native enzyme eluted in three peaks of 129, 245 and 511 kDa, corresponding to dimeric, tetrameric and octameric forms, respectively and fructose 2,6-bisphosphate (Fru 2,6-P2) influenced this aggregation state. Both the glycolytic (forward) and gluconeogenic (reverse) reactions had relative broad pH optima between pH 6.7 and 8.0. The Fru 2,6-P2 saturation curves were hyperbolic with approximate Ka values of 69 and 82 nm for the forward and reverse reactions, respectively. The enzyme showed hyperbolic saturation curves for all its substrates with Km values comparable with that of other plant PFP, i.e. 150, 37, 39 and 460 µM for fructose 6-phosphate, inorganic pyrophosphate, fructose 1,6-bisphosphate and inorganic phosphate, respectively. Sugarcane PFP’s molecular and kinetic characteristics differed slightly from that of other plant PFP in that: (i) Fru 2,6-P2 directly induced the octameric state from the dimeric state; (ii) Fru 2,6-P2 shifted the pH optimum for the forward reaction to a slightly more basic pH; and (iii) Fru 2,6-P2 increased the Vmax for the forward and reverse reactions by similar amounts.
Additional keyword: carbohydrate metabolism.
Acknowledgements
The South African Sugarcane Research Institute, the South African Department of Trade and Industry and Stellenbosch University sponsored this work. We thank Dr N. J. Kruger (Department of Plant Sciences, Oxford, England) for his kind gift of potato PFP-α and -β antisera.
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