Nonidentical subunits of protocatechuate 3,4-dioxygenase

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) has been reported to have a molecular weight of 700,000 and to consist of eight identical subunits, each containing one atom of ferric iron and a substrate binding site. This subunit has now been found to dissociate further into four smaller subunits of two nonidentical types (alpha2beta2), upon sodium dodecyl sulfate gel electrophoresis. The molecular weights of the alpha and beta subunits were estimated to be 22,500 and 25,000, respectively. Isoelectric focusing of the enzyme in 6 M urea revealed that the isoelectric points of the alpha and beta subunits were 5.2 and 9.5, respectively. Separation of the two subunits was achieved by chromatography on sulfopropyl (SP)-Sephadex in 6 M urea after treatment of the enzyme with 8 M urea at 37 degrees C for 6 h. The NH2-terminal sequence of the alpha subunit was determined to be Pro-Ile-Glu-Leu-Leu-Pro-Glu-Thr-Pro-Ser-Glx-Thr-Ala-Gly and that of the beta subunit, Pro-Ala-Gln-Asp-Asn-Ala-Arg-Phe-Val-Ile-Arg-Asx-Arg-Asx. Phenylalanine was found as the COOH-terminal residue of the alpha subunit. However, the COOH terminus of the beta subunit was not detected by any of three methods employed.