Purification and characterization of 5-ketofructose reductase from Erwinia citreus.
5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose.
Schrimsher
J L
JL
Department of Protein Chemistry, Biogen S.A., Geneva, Switzerland.
Wingfield
P T
PT
Bernard
A
A
Mattaliano
R
R
Payton
M A
MA
D000596:Amino Acids / Q000032:analysis; D002237:Carbohydrate Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004885:Erwinia / Q000201:enzymology*; D005632:Fructose / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D013379:Substrate Specificity
D000596:Amino Acids; D005632:Fructose; D002237:Carbohydrate Dehydrogenases; C001504:fructose 5-dehydrogenase (NADP+)