PORPHOBILINOGEN SYNTHASE FROM ESCHERICHIA-COLI IS A ZN(II) METALLOENZYME STIMULATED BY MG(II)

Porphobilinogen synthase (PBGS) is essential to all life forms; in mammals it is definitively established that Zn(II) is required for activity. The literature regarding the metal requirement for PBGS in other animals, plants, and bacteria neither establishes nor disproves a Zn(II) requirement. We have characterized Escherichia coli PBGS and found it to be remarkably similar to bovine PBGS. The similarities include a requirement for Zn(II), inhibition by 1,10-phenanthroline, an exceptional thermal stability, a requirement for free sulfhydryl(s) as shown by sensitivity to modification by methyl methanethiosulfonate, and the presence of tightly bound product on freshly isolated enzyme. Proton-induced X-ray emission analysis shows E. coli PBGS to contain a stoichiometric amount of Zn and no other metals. The most striking similarity between E. coli and bovine PBGS is the 13C NMR spectrum of enzyme-bound [3,5-13C]PBG; the chemical shifts of bound product are identical for both bovine and E. coli PBGS. Minor differences between E. coli PBGS and its mammalian counterpart include Km (ALA) = 1.9 mM, a pH optimum of 7.5-8, and its molar absorbtion coefficient expressed as A0.1% 280 is 0.588. We conclude from these data that E. coli PBGS is a Zn(II)-metalloenzyme and that Zn(II) is required for catalytic activity, and propose that the mammalian and bacterial PBGS function by similar mechanisms. There is one significant difference between E. coli and mammalian PBGS. For E. coli PeGS, Mg(II) causes a twofold stimulation of the Zn(II)-induced E. coli PBGS activity; this effect is not seen for bovine PBGS. The stimulation of activity by Mg(II) mimics the effect of Mg(II) on plant PBGS, although E. coli PBGS does not contain the putative Mg(II) binding site recently revealed by Boese et al. [Q. F. Boese, A. J. Spano, T. Li, and M. P. Timko (1991) J. Biol. Chem. 266, 17060-170661. This work lays the foundation for identification of functional amino acids based on the sequence similarities between PBGS from bacterial, plant, and mammalian sources. © 1993 Academic Press, Inc.