Biochemical characterization of cytosolic fructose-1,6-bisphosphatase from apple (Malus domestica) leaves.
Cytosolic fructose-1,6-bisphosphatase was purified to apparent homogeneity from the leaves of apple, a sorbitol synthesizing species. The enzyme was a homotetramer with a subunit mass of 37 kDa, and was highly specific for fructose 1,6-bisphosphate (F1,6BP) with a Km of 3.1 micro M and a Vmax of 48 units (mg protein)(-1). Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mM and 62 micro M, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+ activated enzyme activity. Fructose 6-phosphate (F6P) was found to be a mixed type inhibitor with a Ki of 0.47 mM. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. AMP was a non-competitive inhibitor for the enzyme. F6P interacted with F2,6BP and AMP in a synergistic way to inhibit the enzyme activity. Dihydroxyacetone phosphate slightly inhibited the enzyme activity in the presence or absence of F2,6BP. Sorbitol increased the susceptibility of the enzyme to the inhibition by high concentrations of F1,6BP. High concentrations of sorbitol in the reaction mixture led to a reduction in the enzyme activity.
Department of Horticulture, Cornell University, Ithaca, NY 14853, USA.
Rui
R
Zhou
Lailiang
L
Cheng
D000249:Adenosine Monophosphate / Q000737:chemistry; D001667:Binding, Competitive / Q000502:physiology; D003600:Cytosol / Q000201:enzymology*; D004099:Dihydroxyacetone Phosphate / Q000737:chemistry; D005632:Fructose / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005635:Fructosediphosphates / Q000737:chemistry* / Q000378:metabolism; D005636:Fructosephosphates / Q000737:chemistry; D008274:Magnesium / Q000378:metabolism; D027845:Malus / Q000201:enzymology* / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology*; D013012:Sorbitol / Q000737:chemistry; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism
D005635:Fructosediphosphates; D005636:Fructosephosphates; D005632:Fructose; D000249:Adenosine Monophosphate; D008345:Manganese; D013012:Sorbitol; D004099:Dihydroxyacetone Phosphate; D013395:Sucrose; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium; C029063:fructose-1,6-diphosphate