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Higgins, W and Miles, EW (1978) Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase. J. Biol. Chem. 253:4648-52


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.




Affinity Labels; Apoenzymes; Binding Sites; Escherichia coli/enzymology; Peptide Fragments; Pyridoxal Phosphate/metabolism; Pyridoxamine/analogs & derivatives; Tryptophan Synthase/antagonists & inhibitors; Tryptophan Synthase/metabolism


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