Purification and properties of uroporphyrinogen I synthase from human erythrocytes. Identification of stable enzyme-substrate intermediates

Uroporphyrinogen I synthase (EC 4.3.1.8) was purified from human erythrocytes more than 42,000-fold with a 25% yield. The purification procedure included a preparative DEAE-cellulose step followed by sequential chromatography on octyl-Sepharose, phenyl-Sepharose, Sephadex G-100, and DEAE-cellulose. The final anion exchange step resolved uroporphyrinogen I synthase activity into five forms which were designated A, B, C, D, and E. These forms also were separated by polyacrylamide disc gel electrophoresis and isoelectric focusing. The least charged forms, A and B, represented about 75% of total activity and appeared homogeneous by sodium dodecyl sulfate and analytical polyacrylamide gel electrophoresis at pH 6.8 and 8.2. The A and B forms had similar physical and kinetic properties including specific activities of about 2,300 units/mg of protein, pH optima of 8.2 K(m) values of ~6 μM, inhibition by sulfhydryl reagents, and identical amino acid compositions. The molecular weights of the A and B activities were 36,000 and 38,000 by gel filtration; under denaturing conditions both A and B forms had a molecular weight of 37,000, indicating that these forms were monomers. [³H]Porphobilinogen was synthesized and used to demonstrate that the multiple forms represented intermediates in the stepwise conversion of the monopyrrole to the tetrapyrrole. When homogeneous A or B enzyme was individually incubated with [³H]porphobilinogen and then subjected to electrophoresis, all five forms were observed; the more anodal bands of activity contained proportionately more radiolabel. These data are consistent with uroporphyrinogen I synthase A being the native enzyme and the B, C, D, and E charge isomers corresponding to the enzyme-substrate (mono-, di-, tri- and tetrapyrrole) intermediates.