The Subunit Structure of Crotonase

Crystalline bovine crotonase has a molecular weight of 164,000 ± 3,000 in phosphate buffer as determined by the method of sedimentation equilibrium in the ultracentrifuge. In contrast, crotonase has a molecular weight of 28,000 ± 500 in 6 m guanidine hydrochloride, or about one-sixth that of the native enzyme. The enzyme contains no disulfide bonds inasmuch as the total number of free thiol groups titrated with 5,5'-dithiobis(2-nitrobenzoate) is equal to the total half-cystine content. Tryptic peptide maps indicate only about one-sixth the expected number of peptide based upon the lysine and arginine content of the enzyme. End group analysis shows serine at the NH 2 terminus and aspartic acid at the COOH terminus of the molecule. These data suggest that crotonase is composed of 6 subunit polypeptide chains, with each chain containing about 260 residues in an identical or very similar sequence, and that the 6 chains are combined through noncovalent interactions to form the native enzyme.