PURIFICATION AND CHARACTERIZATION OF FRUCTOKINASE FROM DEVELOPING TOMATO (LYCOPERSICON-ESCULENTUM MILL) FRUITS

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fol d purification and two isoenzymes designated fructokinase I and II (FK I and FKII) were separated by anion-exchange chromatography. Using sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose -12 indicated that for both fructokinases the functional form is a dim er. Two dimensional isoelectric focusing/SDS-PAGE combined with immuno blotting showed that FKI has two components with isoelectric points (p Is) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that t he components of FKI match the more alkaline components of FKII. The a ctivity of both fructokinases increased with increasing pH to around 8 .0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K-m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants ( K-i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K-m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05-0 .2 mM), fructose exhibited noncompetitive inhibition with respect to A TP for both fructokinases. This inhibition pattern changed to uncompet itive when higher fructose concentrations (0.5-10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding fi rst. Inhibition by ADP was also affected by the fructose concentration s. At 0.5 mM fructose, FKI showed noncompetitive inhibition by ADP wit h respect to ATP and this inhibition changed to uncompetitive when 3 m M fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both f ructokinases suggest that this enzyme might have a relevant role in ca rbon metabolism during tomato fruit development.