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 (AL A) to form porphobilinogen. There are at least four types of PEGS, cat egorized according to metal ion usage. The PBGS from Bradyrhizobium ja ponicum requires Mg(II) in catalytic metal site A; has an allosteric M g(II) in metal site C, and also contains an activating monovalent cati on binding site [Petrovich et al, (1996) J. Blob. Chem. 271, 8692-8699 ]. C-13 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 C-13 NMR che mical shifts of enzyme-bound product demonstrate that the chemical env ironment of porphobilinogen bound to B. japonicum PEGS is different fr om 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 interac tion between Mn-A and product at the active site. Prior characterizati on of the enzyme defined conditions in which the divalent cation occup ies either the A or the C site. Mimicking these conditions allows Mn(I I) EPR observation of either Mn-C or Mn-A, The EPR spectrum of Mne is significantly broader and less intense than ''free'' Mn(II), but relat ively featureless. The EPR spectrum of Mn-A is broader still and more asymmetric than Mnc. The EPR data indicate that the coordination spher es of the two metals are different.