Abstract
Human L-glutamine: D-fructose-6-phosphate amidotransferase (Gfat1), a recognized target in type 2 diabetes complications, was expressed in Sf9 insect cells with an internal His(6)-tag and purified to homogenity. Two different microplate assays that quantify, respectively D-glucosamine-6-phosphate and L-glutamate were used to analyze the enzyme kinetic properties. The recombinant human L-glutamine: D-fructose-6-phosphate amidotransferase isoform 1 exhibits Michaelis parameters K(m)(Fru-6P)=0.98 mM and K(m)(Gln)=0.84 mM which are similar to the values reported for the same enzyme from different sources. The stimulation of hydrolysis of the alternate substrate L-glutamine para-nitroanilide by D-fructose-6P (Fru-6P) afforded a K(d) of 5 microM for Fru-6P.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Cells, Cultured
-
Circular Dichroism
-
Cloning, Molecular
-
Enzyme Activation
-
Fructosephosphates / chemistry
-
Glutamic Acid / chemistry
-
Glutaminase / chemistry
-
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / biosynthesis*
-
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / chemistry
-
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / isolation & purification*
-
Histidine / chemistry*
-
Humans
-
Hydrolysis
-
Insecta / cytology
-
Insecta / metabolism
-
Kinetics
-
Protein Structure, Tertiary
-
Recombinant Proteins / biosynthesis*
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / isolation & purification*
Substances
-
Fructosephosphates
-
Recombinant Proteins
-
Glutamic Acid
-
Histidine
-
fructose-6-phosphate
-
GFPT1 protein, human
-
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
-
Glutaminase