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) clo ned from a tomato fruit cDNA library has been expressed in Escherichia coli. The recombinant protein was purified 159-fold to greater than 9 9% purity, based on SDS-PAGE analysis. The subunit molecular mass is e stimated to be 35 kDa and the nondissociated molecular mass is 72.4 kD a, 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 grou ped peptides with pI values ranging from 5.66 to 6.17. Biochemical cha racterization of the purified recombinant enzyme shows properties esse ntially identical to those of the native fructokinase purified from yo ung 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 concentr ation is greater than 0.5 mM (K-i = 3.0 mM). ATP is the preferred phos phate donor (K-m = 0.13 mM and V-max/K-m = 212), followed by GTP (K-m = 0.45 mM and V-max/K-m = 76) and UTP (K-m = 1.68 mM and V-max/K-m = 2 0), but V-max values are slightly greater with GTP and UTP. Product in hibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration [K-i (ADP) = 0.41 mM with 0.5 m M fructose and decreased to 0.12 mM with 3 mM fructose]. Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fru ctose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK. (C) 1997 Academic Press.