Affinity Labeling of Escherichia Coli Glucosamine‐6‐Phosphate Synthase with a Fructose 6‐Phosphate Analog

Abstract

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the y-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k,,,,, = 0.60 -C 0.05 min-I , K,,, = 1.40 +-0.20 mM) by anhydro-l,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of sidechain carboxylates of GIu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne

Pathways for the utilization of N‐acetyl‐galactosamine and galactosamine in Escherichia coli

“…GlmS) thought to be responsible for the aldose–ketose isomerase activity of these enzymes. AgaS lacks, however, the C‐terminal or amidotransferase domain normally present in this class of synthases (Denisot et al ., 1991; Leriche et al ., 1997). Overproduction of AgaS did not complement GlmS negative mutants of either E. coli C or K‐12 (our unpublished results).…”

Novel Enzymatic Mechanisms in the Biosynthesis of Unusual Sugars

“…The deprotonation is pro-R-stereospecific, as the majority of the isotope label was washed out when (1R)-D-[1-3 H]fructose-6-phosphate was incubated with the enzyme. Incubation of this compound, which should bind at the synthe-tase domain, results in the covalent modification of Cys1 in the glutaminase domaine [119]. Since exogenous NH 3 cannot substitute for glutamine in the glucosamine-6phosphate synthetase-catalyzed reaction, the glutamine hydrolyzing activity and sugar aminating activity of this enzyme must be tightly coupled.…”

Inhibition of Escherichia coliGlucosamine-6-phosphate Synthase by Reactive Intermediate Analogues

“…NMR spectra ( 13 C, 31 P) were obtained using a Bruker AC 250F spectrometer. Chemical shifts (␦) for 13 C and 31 P spectra are reported relative to the deuterium lock signal and external H 3 PO 4 (85% w/v in D 2 O), respectively. Elemental analyses were performed by Canadian Microanalytical Service Ltd., B. C.…”

“…Fractions containing dGlcol-6-P were combined and lyophilized yielding a hygroscopic glassy powder (0.1953 g, 78% yield). 13 (22,23). The oxime was then converted to its corresponding 5-phosphate using hexokinase-catalyzed phosphorylation as described by Finch and Merchant (20).…”