Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase

Abstract

Listen

Translate article

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.