Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.
Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.
da-Silva
W S
WS
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, RJ, Brasil.
Rezende
G L
GL
Galina
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005631:Fructokinases / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006601:Hexoses / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology*
D004791:Enzyme Inhibitors; D006601:Hexoses; D005632:Fructose; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005631:Fructokinases; D006593:Hexokinase; C046173:fructokinase; D005947:Glucose