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

The synthesis of tetrapyrroles is essential to all phyla. Porphobilino gen synthase (PEGS) is a zinc metalloenzyme that catalyzes the formati on of porphobilinogen, the monopyrrole precursor of all biological tet rapyrroles. The enzyme from various organisms shows considerable seque nce 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 stimula tory Mg(II) is called Mg-C. Mg-C is nor required for activity and is d istinct 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 th e substrate 5-aminolevulinic acid (ALA) and the V-max are altered by t he presence of Mg-C; Mg(II) causes the K-m to drop from similar to 3 t o 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 activa tion by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mg-C site is pro bably saturated under physiological conditions. Mn(II) is a good subst itute for Mg-C, giving a comparable increase in catalytic activity. Co nsequently, Nln(LI) has been used as an EPR active probe of the Mg-C b inding 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 bindin g site with rhombic symmetry and are consistent with oxygen and/or nit rogen ligands. The addition of unlabeled or 1-C-13-labeled ALA does no t significantly affect the Mn(II) EPR spectra. The hl,oc binding sites apparently are distant from each other and also distant from the acti ve sites. The eight equivalent Mg-C's of PBGS(E. coli) are in sharp co ntrast to the four active sites. Mg-C has a profound effect on the qua ternary structure of the protein. PBGS(E. coli) octamers dissociate in to 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. T wo-dimensional electrophoresis experiments demonstrate that the protei n can reassemble within the gel to form octamers upon incubation with Mg(II) and/or ALA. Catalytic activity is observed for all species with in the gel. Since assay conditions promote octamer formation, it is no t possible to determine whether smaller species are active.