The interaction of Escherichia coli tryptophanase with various amino acids and their analogs. Active site mapping.:Active Site Mapping

Tryptophanase [TPase, L-tryptophan indole-lyase, EC 4. 1. 99. 1] from Escherichia coli B was studied extensively with respect to its interaction with various amino acids and their analogs. Several new substrates were found; these include β-chloro-L-alanine, L-cysteine sulfinate, S-benzyl-L-cysteine, O-benzyl-L-serine, L-threonine, L-allothreonine, and the two β-methyl-L-tryptophans. Formation of S-hydroxyethyl-L-cysteine from L-serine and 2-mercaptoethanol was also demonstrated. Most amino acids tested, including these substrates, were competitive inhibitors of the TPase reaction with L-tryptophan as the substrate. The K₁ values differed so markedly among various amino acids that it was possible to draw conclusions concerning the topography of the active site of TPase. The indole ring or other electronegative group on the β-carbon atom, together with a carboxyl group on the α-carbon atom, is necessary for oriented binding to the active site of TPase; a free α-amino group is also required for substrate activity, but does not contribute greatly to the binding interactions.<br> Most amino acids and their analogs induced an absorption peak near 500nm, previously assigned to quinoidal structure formed by labilization of the α-proton of the enzyme-bound Schiff's base between PLP and amino acid. Amino acids that have an asymmetric carbon at the β-position (L-threonine, L-allothreonine, L-isoleucine, , β-methyl-L-tryptophan) and some other amino acids (L-valine, phenylglycine, glycine) did not evoke this 500nm peak.<br> Bromopyruvate proved to be a very potent inactivator of TPase. Pyridoxal 5'-phosphate completely prevented this inactivation, but inhibitor amino acids did not. One SH group per subunit of TPase was blocked by treatment with bromopyruvate. The cysteine residue that reacts is probably located in the peptide T-18-1 isolated by Kagamiyama et al. [J. Biol. Chem. 247, 1571-1575 (1972)]. TPCK inactivated TPase in a manner similar to bromopyruvate.