Characterization of the role of the stimulatory magnesium of Escherichia coli porphobilinogen synthase.
The synthesis of tetrapyrroles is essential to all phyla. Porphobilinogen synthase (PBGS) 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 PBGS are activated by magnesium, a property that is absent from mammalian PBGS. This stimulatory Mg(II) is called Mgc. Mgc is not required for activity and is distinct from the two zinc ions (ZnA and ZnB) common to mammalian and E. coli PBGS (PBGSE.coli). For PBGSE.coli, both the Km for the substrate 5-aminolevulinic acid (ALA) and the Vmax are altered by the presence of Mgc; Mg(II) causes the Km to drop from approximately 3 to 0.30 mM and the maximum specific activity to increase from 23 to 50 mumol h-1 mg-1. Mgc also causes the saturating concentration of the required Zn(II) to decrease from 0.1 mM to 10 microM. Maximal activation by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mgc site is probably saturated under physiological conditions. Mn(II) is a good substitute for Mgc, giving a comparable increase in catalytic activity. Consequently, Mn(II) has been used as an EPR active probe of the Mgc 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 1-13C-labeled ALA does not significantly affect the Mn(II) EPR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)
Jaffe
E K
EK
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Ali
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Mitchell
L W
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Taylor
K M
KM
Volin
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Markham
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GD
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000502:physiology*; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D000623:Porphobilinogen Synthase / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000502:physiology
D008345:Manganese; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc