Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Abstract

Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K m 0.2-1 mM). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K m for Fru-6-P of 7 M, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K d of 2 M was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K i measured of 6 M. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDPGlcNAc, was competitive with a K i of 4 M. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.

N-Acetylglucosamine Inhibits T-helper 1 (Th1)/T-helper 17 (Th17) Cell Responses and Treats Experimental Autoimmune Encephalomyelitis

“…Indeed, our previous in vitro studies revealed that increasing concentrations of GlcN initially increased but then reduced N-glycan branching (29). The latter likely arises from GlcN 6-phosphate acting as a potent inhibitor of glutamine fructose-6-phosphate amidotransferase, the ratelimiting enzyme shunting fructose 6-phosphate into the hexosamine pathway (41). In contrast to GlcN, increasing GlcNAc concentrations in vitro is observed to only enhance N-glycan branching.…”

Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

“…Although GlcN6P synthase and GlcN6P deaminase catalyze similar reactions, there is no relation between the primary structures of these two enzymes. There have been many studies on GlcN6P synthase (3,5,13,24) and GlcN6P deaminase (1,8,(15)(16)(17)20) from Eucarya and Bacteria due to the importance of these enzymes in the regulation of amino sugar metabolism. In contrast, corresponding enzymes from Archaea have not been reported so far.…”

Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase

“…Indeed, GlmS synthesis is also activated through discoordinate expression of the glmUS operon by a conserved small RNA in several Gram-negative bacteria, including E. coli (43). In contrast, in Gram-positive bacteria, post-transcriptional regulation is performed by the glmS ribozyme (44,45), whereas a more classical post-translational regulation resulting from UDP feedback inhibition or enzyme phosphorylation was reported for the eukaryotic enzymes from C. albicans (34,46), Drosophila (47), and humans (48,49).…”