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 format ion of glucosamine 6-phosphate from fructose 6-phosphate using the gam ma-amide functionality of glutamine as the nitrogen source. In the abs ence of glutamine, GlcNP-synthase was recently found to catalyze the f ormation of glucose 6-phosphate corresponding to a phosphoglucoisomera se-like activity. Here we report active-site directed, irreversible in hibition of Escherichia coli GlcNP-synthase (k(inact) = 0.60+/-0.05 mi n(-1), K-irr = 1.40+/- 0.20 mM) by anhydro-1,2-hexitol 6-phosphates pr eviously known as irreversible inhibitors of phosphoglucoisomerase. En zyme inactivation with the tritiated affinity label, followed by trypt ic digestion and purification of the radioactive fragments, allowed id entification 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 C ys300 of the fructose-6-phosphate-binding site on the epoxide function ality of the inhibitor. The major peptide corresponds to derivatizatio n of the N-terminal cysteine from the glutamine-binding site by the in hibitor. These results provide evidence for the close proximity of glu tamine and fructose-6-phosphate-binding sites recently suggested by Be arne.