Expression and purification of active human internal His(6)-tagged L-glutamine: D-Fructose-6P amidotransferase I.
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.
Richez
Céline
C
Institut de Chimie des Substances Naturelles, ICSN-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Boetzel
Joachim
J
Floquet
Nicolas
N
Koteshwar
Kinnera
K
Stevens
Julie
J
Badet
Bernard
B
Badet-Denisot
Marie-Ange
MA
D000818:Animals; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005636:Fructosephosphates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D005972:Glutaminase / Q000737:chemistry; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D005636:Fructosephosphates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; C027618:fructose-6-phosphate; C497511:GFPT1 protein, human; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005972:Glutaminase