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Petrovich RM , Jaffe EK
Magnetic resonance studies on the active site and metal centers of Bradyrhizobium japonicum porphobilinogen synthase
Biochemistry. 1997 Oct 28;36(43) :13421-13427
PMID: ISI:A1997YD64900035   
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Abstract
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 PEGS, 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. 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 chemical shifts of enzyme-bound product demonstrate that the chemical environment of porphobilinogen bound to B. japonicum PEGS 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 Mn-A 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 Mn-C or Mn-A, The EPR spectrum of Mne is significantly broader and less intense than ''free'' Mn(II), but relatively featureless. The EPR spectrum of Mn-A is broader still and more asymmetric than Mnc. The EPR data indicate that the coordination spheres of the two metals are different.
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Times Cited: 6 English Article YD649 BIOCHEMISTRY-USA