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- 6 P = 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 μM for Fru-6P.
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