Chemical modification studies of the active site of glucosamine-6-phosphate synthase from baker's yeast.

Glucosamine-6-phosphate synthase from baker's yeast has been purified 100-fold with a final recovery of 70%. The purification procedure involved thiol-affinity chromatography. Chemical modification studies of the enzyme revealed the presence of cysteine, Glu/Asp-carboxyl and probably histidine at the glutamine binding site and, on the other hand, arginine and probably another histidine at the D-fructose 6-phosphate binding site. A few glutamine analogs, including 6-diazo-5-oxo-L-norleucine (DON), anticapsin and N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), were shown to inactivate the enzyme in a time- and concentration-dependent manner. Anticapsin, the most active in the series, exhibited an inactivation constant, Kinact, of 9.5.10(-6) M.