5-CHLORO[1,4-C-13]LEVULINIC ACID MODIFICATION OF MAMMALIAN AND BACTERIAL PORPHOBILINOGEN SYNTHASE SUGGESTS AN ACTIVE-SITE CONTAINING 2 ZN(II)

5-Chloro[1,4-C-13]levulinic acid ([1,4-C-13]CLA) is an active site-dir ected inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrica lly condenses two molecules of 5-aminolevulinic acid (ALA) which are c alled A-side ALA and P-side ALA in reference to their fates as the ace tyl and propionyl halves of the product. [1,4-C-13]CLA modifies bovine PBGS at the A-side ALA binding site. The C-4 chemical shift indicates an intact keto moiety; the C-1 chemical shift indicates a deprotonate d carboxyl group. In contrast, [1,4-C-13]CLA modification of Escherich ia coil PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C-1 chemical shifts indicate substantially depro tonated carboxylic acid groups. For one of four observed forms of [1,4 -C-13]CLA-modified E. coli PBGS, an analog of the P-side Schiff base i s found. Bovine and E. coli PBGS contain two different zincs, Zn-A and Zn-B. Past results placed Zn-A near A-side ALA. [1,4-C-13]CLA modifie s E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine c luster implicated in binding Zn-B. This result places Zn-B near P-side ALA. E. coli PBGS binds a third type of divalent metal, Mg-C or Mn-C, which is found to have no significant effect on the C-13 NMR spectrum of the [1,4-C-13]CLA-modified protein.