CHARACTERIZATION OF THE ROLE OF THE STIMULATORY MAGNESIUM OF ESCHERICHIA-COLI PORPHOBILINOGEN SYNTHASE

The synthesis of tetrapyrroles is essential to all phyla. Porphobilinogen synthase (PEGS) is a zinc metalloenzyme that catalyzes the formation of porphobilinogen, the monopyrrole precursor of all biological tetrapyrroles. The enzyme from various organisms shows considerable sequence conservation, suggesting a common fold, quaternary structure, and catalytic mechanism. Escherichia coli and plant PEGS are activated by magnesium, a property that is absent from mammalian PEGS. This stimulatory Mg(II) is called Mg-C. Mg-C is nor required for activity and is distinct from the two zinc ions (Zn-A and Zn-B) common to mammalian and E. coli PBGS (PBGS(E. coli) ). For PBGS(E. coli,) both the K-m for the substrate 5-aminolevulinic acid (ALA) and the V-max are altered by the presence of Mg-C; Mg(II) causes the K-m to drop from similar to 3 to 0.30 mM and the maximum specific activity to increase from 23 to 50 mu mol h(-1) mg(-1). Mg-C also causes the saturating concentration of the required Zn(II) to decrease from 0.1 mM to 10 mu M. Maximal activation by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mg-C site is probably saturated under physiological conditions. Mn(II) is a good substitute for Mg-C, giving a comparable increase in catalytic activity. Consequently, Nln(LI) has been used as an EPR active probe of the Mg-C binding site. Mn(II) binds at a stoichiometry of eight ions per enzyme octamer. The X- and Q-band EPR spectra reflect a single type of binding site with rhombic symmetry and are consistent with oxygen and/or nitrogen ligands. The addition of unlabeled or 1-C-13-labeled ALA does not significantly affect the Mn(II) EPR spectra. The hl,oc binding sites apparently are distant from each other and also distant from the active sites. The eight equivalent Mg-C's of PBGS(E. coli) are in sharp contrast to the four active sites. Mg-C has a profound effect on the quaternary structure of the protein. PBGS(E. coli) octamers dissociate into smaller species during native gel electrophoresis. Preincubation of the protein in EDTA potentiates the dissociation, while preincubation of the protein in Mg(II) or Mn(II) and/or ALA hinders dissociation. Two-dimensional electrophoresis experiments demonstrate that the protein can reassemble within the gel to form octamers upon incubation with Mg(II) and/or ALA. Catalytic activity is observed for all species within the gel. Since assay conditions promote octamer formation, it is not possible to determine whether smaller species are active.