The Chemical Structure of Tryptophanase from Escherichia coli: III. ISOLATION AND AMINO ACID SEQUENCE OF THE TRYPTIC PEPTIDES

Abstract

Abstract Sodium borohydride-reduced, carboxymethylated holotryptophanase was digested with trypsin, and the resulting peptides were purified by column chromatography on basic and acidic ion exchange resins, and by paper chromatography and electrophoresis. The isolation and sequence determinations of 13 peptides, including the NH2- and COOH-terminal peptides, those containing carboxymethylcysteine or tryptophan, and that containing the pyridoxyllysine residue (Lys(Pxy)) are reported here, together with a summary of the sequences of a total of 54 tryptic peptides present in the digest. The NH2-terminal peptide, Met-Glu-Asn-Phe-Lys, and the COOH-terminal peptide, Leu-Lys-Glu-Val, were isolated, and an extended sequence at the COOH-terminal end of tryptophanase was proposed to be His-Thr-Phe-Ala-Lys-Leu-Lys-Glu-Val on the basis of carboxypeptidase A experiments and the sequence of an isolated pentapeptide. The amino acid sequence of an extended portion of the peptide chain around the reduced pyridoxal 5-phosphate residue was determined to be Tyr-Ala-Asp-Met-Leu-Ala-Met-SerAla-Lys-Lys(Pxy)-Asp-Ala-Met-Val-Pro-Met-Gly-Gly-Leu-Leu-Cys(Cm)-Met-Lys. Tryptophanase contains 2 tryptophan and 6 cystine residues per subunit of 55,000 molecular weight, and two distinct tryptophan peptides and five distinct carboxymethylcysteine peptides were isolated and sequenced. The studies thus support the view that this subunit contains a single peptide chain. Of a total of about 474 amino acid residues per subunit, 411 were recovered in the 54 tryptic peptides so far studied.