TI - Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 ( GFAT2 ) by cAMP -dependent protein kinase increases the enzyme activity . AB - A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase ( GFAT ) was termed GFAT2 on the basis of this similarity . The mouse GFAT2 cDNA was cloned , and the protein was expressed with either an N-terminal glutathione S-transferase or His tag . The purified protein expressed in mammalian cells had GFAT activity . The Km values for the two substrates of reaction , fructose 6-phosphate and glutamine , were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine , which are within the ranges determined for GFAT1 . The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1 , whereas the serine at 235 in GFAT1 was not present in GFAT2 . Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of c-AMP -dependent protein kinase ( PKA ) inhibits its activity . Like GFAT1 , GFAT2 was phosphorylated by PKA , but GFAT2 activity increased approximately 2.2-fold by this modification . When serine 202 of GFAT2 was mutated to an alanine , the enzyme not only became resistant to phosphorylation , but also the increase in activity in response to PKA also was blocked . These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA . GFAT2 was modestly inhibited ( 15% ) by UDP-GlcNAc but not through detectable O-glycosylation . GFAT2 is , therefore , an isoenzyme of GFAT1 , but its regulation by cAMP is the opposite , allowing differential regulation of the hexosamine pathway in specialized tissues .