title: Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase.
abstract: We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.
mesh_terms: D000345:Affinity Labels; D001051:Apoenzymes; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives*; D014367:Tryptophan Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001051:Apoenzymes; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D014367:Tryptophan Synthase