Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.
Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the gamma-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(inact) = 0.60 +/- 0.05 min(-1), Kirr = 1.40 +/- 0.20 mM) by anhydro-1,2-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 side-chain carboxylates of Glu255 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 [Bearne, S. L. (1996) J. Biol. Chem. 271, 3052-3057].
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Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, Gif-sur-Yvette, France.
Badet-Denisot
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Badet
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D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006600:Hexosephosphates / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014357:Trypsin / Q000378:metabolism
C002826:1,2-anhydro-hexitol-6-phosphate; D000345:Affinity Labels; D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D006600:Hexosephosphates; D005973:Glutamine; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D014357:Trypsin; D003545:Cysteine