Magnetic resonance studies on the active site and metal centers of Bradyrhizobium japonicum porphobilinogen synthase.
Porphobilinogen synthase (PBGS) is a metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen. There are at least four types of PBGS, categorized according to metal ion usage. The PBGS from Bradyrhizobium japonicum requires Mg(II) in catalytic metal site A, has an allosteric Mg(II) in metal site C, and also contains an activating monovalent cation binding site [Petrovich et al. (1996) J. Biol. Chem. 271, 8692-8699]. 13C NMR and Mn(II) EPR have been used to probe the active site and Mg(II) binding sites of this 310 000 dalton protein. The 13C NMR chemical shifts of enzyme-bound product demonstrate that the chemical environment of porphobilinogen bound to B. japonicum PBGS is different from that of PBGS which contains Zn(II) rather than Mg(II) at the active site. Use of Mn(II) in place of Mg(II) broadens the NMR resonances of enzyme-bound porphobilinogen, providing evidence for a direct interaction between MnA and product at the active site. Prior characterization of the enzyme defined conditions in which the divalent cation occupies either the A or the C site. Mimicking these conditions allows Mn(II) EPR observation of either MnC or MnA. The EPR spectrum of MnC is significantly broader and less intense than "free" Mn(II), but relatively featureless. The EPR spectrum of MnA is broader still and more asymmetric than MnC. The EPR data indicate that the coordination spheres of the two metals are different.
Petrovich
R M
RM
Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA.
Jaffe
E K
EK
D001665:Binding Sites / Q000187:drug effects; D002250:Carbon Radioisotopes; D004492:Edetic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D011162:Porphobilinogen / Q000737:chemistry / Q000378:metabolism; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000378:metabolism*; D012230:Rhizobiaceae / Q000201:enzymology*
D002250:Carbon Radioisotopes; D008345:Manganese; D011162:Porphobilinogen; D004492:Edetic Acid; D000623:Porphobilinogen Synthase; D008274:Magnesium