Escherichia coli tryptophan indole-lyase (Trpase) is a pyridoxal 5'-phosphate(pyridoxal-P)-dependent enzyme which catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal-P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20-100-fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal-P, and a shift in the lambda(max) from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal-P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50-fold for native enzyme, but the K(A) for K+ is unaffected. Both the native and trypsin-nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid-scanning stopped-flow experiments. However, the rate constant for quinonoid intermediate formation from L-tryptophan is reduced by at least 400-fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of L-alanine and S-ethyl-L-cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray-ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active-site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate alpha-carboxylate-binding site, and His463, are located in the sequence between Lys406 and the C-terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.