Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.
Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.
Petreikov
M
M
Department of Vegetable Crops, Agricultural Research Organization, Volcani Center, 50250, Bet Dagan, Israel.
Dai
N
N
Granot
D
D
Schaffer
A A
AA
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005631:Fructokinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005632:Fructose; D005631:Fructokinases; C046173:fructokinase