Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli.
Fructokinase (FK; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) cloned from a tomato fruit cDNA library has been expressed in Escherichia coli. The recombinant protein was purified 159-fold to greater than 99% purity, based on SDS-PAGE analysis. The subunit molecular mass is estimated to be 35 kDa and the nondissociated molecular mass is 72.4 kDa, indicating that the functional form is a dimer. Two-dimensional IEF/SDS-PAGE analyses combined with immunodetection show that both native and recombinant proteins exhibit the same pattern of six closely grouped peptides with pI values ranging from 5.66 to 6.17. Biochemical characterization of the purified recombinant enzyme shows properties essentially identical to those of the native fructokinase purified from young tomato fruit: the pH optimum is 8.0, the K(m) for fructose is 0.22 mM, and severe substrate inhibition is observed when fructose concentration is greater than 0.5 mM (Ki = 3.0 mM). ATP is the preferred phosphate donor (K(m) = 0.13 mM and Vmax/K(m) = 212), followed by GTP (K(m) = 0.45 mM and Vmax/K(m) = 76) and UTP (K(m) = 1.68 mM and Vmax/K(m) = 20), but Vmax values are slightly greater with GTP and UTP. Product inhibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration [Ki (ADP) = 0.41 mM with 0.5 mM fructose and decreased to 0.12 mM with 3 mM fructose]. Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fructose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK.
Martinez-Barajas
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Biochemistry Department, University of Missouri, Columbia 65211, USA. bcemati@muccmail.missouri.edu
Krohn
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BM
Stark
D M
DM
Randall
D D
DD
D003001:Cloning, Molecular; D004926:Escherichia coli; D005631:Fructokinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D005631:Fructokinases; C046173:fructokinase