The Subunit Structure of Tryptophanase: II. A CORRELATION OF ULTRACENTRIFUGAL AND CHEMICAL STUDIES

Abstract

Abstract At high pH values or in the presence of sodium dodecyl sulfate, 5 m guanidinium chloride, or 8 m urea, apotryptophanase (mol wt 220,000) dissociates into four subunits of molecular weight near 55,000. Evidence is presented as follows that each of these subunits contains two apparently identical peptide chains joined through a disulfide linkage. (a) Reduced, carboxymethylated and performate-oxidized apoptrytophanase both show molecular weights of 28,000 to 30,000; apotryptophanase in 5 m guanidinium chloride shows a similar molecular weight only after treatment with dithiothreitol at alkaline pH values. (b) Analyses for cysteic acid in hydrolysates of performate-oxidized apotryptophanase and for carboxymethylcysteine in reduced and carboxymethylated apotryptophanase both show the presence of 24 half-cystine residues per molecule, in agreement with the 24 —SH groups titratable with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the dithiothreitol-treated, denatured protein. Only 16 —SH groups are found by DTNB titration or by carboxymethylation if reduction with dithiothreitol is omitted. These results indicate that apotryptophanase contains 16 free —SH groups and 4 disulfide bridges per molecule (mol wt 220,000), corresponding to 1 disulfide bridge per subunit. Of the 16 free —SH groups, only 8 react with DTNB in the native enzyme, and these are essential for enzymatic activity. (c) Dinitrophenylation yielded 7.2 moles (corrected) of dinitrophenylmethionine, and catalytic hydrazinolysis yielded 8.4 moles (corrected) of valine, per 220,000 g of apotryptophanase. Digestion of denatured apotryptophanase with carboxypeptidase A yielded 7.3 moles of valine per 220,000 g. These values indicate the presence of eight peptide chains with identical NH2-terminal (methionine) and COOH-terminal (valine) amino acids. (d) Peptide maps of reduced, carboxymethylated apotryptophanase following tryptic digestion showed the presence of 34 to 36 ninhydrin-reactive spots, 15 spots positive to Sakaguchi's reagent, and 1 tryptophan-containing spot. Acrylamide gel electrophoresis of cyanogen bromide-treated apotryptophanase showed nine zones. These values are within experimental error of those expected from quantitative amino acid analyses on the assumption that eight identical peptide chains are present per 220,000 g of tryptophanase (i.e. 31 eq of residues of lysine plus arginine, 14 of arginine, 1 of tryptophan, and 8 of methionine per 27,500 g of apotryptophanase). Since tetrameric native apotryptophanase contains only four binding sites for pyridoxal-P per molecule, the coenzyme must bind to only one of the two peptide chains comprising each monomer. The distribution of peptides in maps of tryptic digests of apoenzyme and reduced holoenzyme supports this supposition.