5-Chloro[1,4-13C]levulinic acid ([1,4-13C]CLA) is an active site-directed inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrically condenses two molecules of 5-aminolevulinic acid (ALA) which are called A-side ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. [1,4-13C]CLA modifies bovine PBGS at the A-side ALA binding site. The C4 chemical shift indicates an intact keto moiety; the C1 chemical shift indicates a deprotonated carboxyl group. In contrast, [1,4-13C]CLA modification of Escherichia coli PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C1 chemical shifts indicate substantially deprotonated carboxylic acid groups. For one of four observed forms of [1,4-13C]CLA-modified E. coli PBGS, an analog of the P-site Schiff base is found. Bovine and E. coli PBGS contain two different zincs, ZnA and ZnB. Past results placed ZnA near A-side ALA. [1,4-13C]CLA modifies E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine cluster implicated in binding ZnB. This result places ZnB near P-side ALA. E. coli PBGS binds a third type of divalent metal, MgC or MnC, which is found to have no significant effect on the 13C NMR spectrum of the [1,4-13C]CLA-modified protein.