Identification of amino acid residues modified by pyridoxal 5'-phosphate in Escherichia coli glutamine synthetase.
Chemical modification studies with pyridoxal 5'-phosphate have indicated that lysine(s) appear to be at or near the active site of Escherichia coli glutamine synthetase (Colanduoni, J., and Villafranca, J. J. (1985) J. Biol. Chem. 260, 15042-15050; Whitley, E. J., Jr., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). Enzyme samples were prepared that contained approximately 1, approximately 2, and approximately 3 pyridoxamine 5'-phosphate residues/50,000-Da monomer; the activity of each sample was 100, 25, and 14% of the activity of unmodified enzyme, respectively. Cyanogen bromide cleavage of each enzyme sample was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their absorbance at 320 nm. These isolated peptides were analyzed for amino acid composition and sequenced. The N terminus of the protein (a serine residue) was modified by pyridoxal 5'-phosphate at a stoichiometry of approximately 1/50,000 Da and this modified enzyme had full catalytic activity. Beyond a stoichiometry of approximately 1, lysines 383 and 352 reacted with pyridoxal 5'-phosphate and each modification results in a partial loss of activity. When various combinations of substrates and substrate analogs (ADP/Pi or L-methionine-SR-sulfoximine phosphate/ADP) were used to protect the enzyme from modification, Lys-352 was protected from modification indicating that this residue is at the active site. Under all experimental conditions employed, Lys-47, which reacts with the ATP analog 5'-p-fluorosulfonylbenzoyl-adenosine does not react with pyridoxal 5'-phosphate.
Di Ianni
C L
CL
Department of Chemistry, Pennsylvania State University, University Park 16802.
Villafranca
J J
JJ
D000595:Amino Acid Sequence; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism
D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D005974:Glutamate-Ammonia Ligase; D003488:Cyanogen Bromide; C010627:pyridoxamine phosphate