The thioester (5) was found to inactivate the enzyme porphobilinogen synthase. Replacement of the ketone functionality in aminolevulinic acid (ALA), shown to be involved in Schiff-base linkage with an active site lysine residue, by a thioester moiety was found to result in loss of enzyme activity. A plausible mode of inactivation is proposed. Porphobilinogen synthase (PBGS, ALA-dehydratase) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA, 1) to give porphobilinogen (PBG, 2), the heterocyclic precursor of all naturally occurring porphyrin and corrin cofactors. 1 The native enzyme in E. coli is an octamer of molecular weight 280 kDa (subunit: 35 kDa) and requires Zn(II) for catalysis. 2 The two molecules of ALA come to reside in the final product in an asymmetric fashion, one giving rise to the acetate (A) side chain and the other forming the propionate (P) side chain of porphobilinogen (Fig. 1). The proposed enzymatic mechanism involves formation of a Schiff-base intermediate between an active site lysine 3 and the C-4 carbonyl group of one of the ALA molecules denoted as P-side ALA since the propionate side chain of the product is derived from it. Subsequently the free amino group of the ALA-Schiff base intermediate undergoes Schiff-base formation with the second ALA molecule, denoted as the A-side ALA. Subsequent reactions lead to the formation of PBG (Fig. 2). 5-Chlorolevulinic acid was introduced as an inactivator of the enzyme by Seehra and Jordan. 4 Further work carried out by Jaffe and co-workers showed that the α-chloroketone functionality labelled an active site cysteine believed to be a ligand for the catalytic zinc. 5 Heterogeneous modification of the enzyme was observed as evidenced by the fact that at least two different cysteines were labelled. We now report the mechanism-based inactivation of the enzyme by using a modified substrate analog approach. Heteroatom modification of the C-3 methylene group of the substrate to produce the ester 4 and thioester 5 results in the generation of a potential scissile C-X (X=O, S) bond while minimising structural deviation. If the active site lysine involved in Schiff-base formation carried out nucleophilic attack at the ester or thioester functionality this could result in inactivation of the enzyme as depicted in Fig. 3.