Possible Involvement of Lys603 from Escherichia Coli Glucosamine-6-phosphate Synthase in the Binding of Its Substrate Fructose 6-phosphate

Pyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with [14C]-cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxal-5'-phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4 degrees C of a enzyme-fructose-6-phosphate complex in a 1:1 molar ratio. Treatment of the enzyme-[14C]fructose-6-phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6-phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C-labelled peptides resulted from the decomposition of an unstable alpha,alpha'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.