Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of the cysteine moiety of 4'-phosphopantothenoylcysteine (PPC) to form 4'-phosphopantetheine (PPantSH); this reaction forms part of the biosynthesis of coenzyme A. The enzyme is a member of the larger family of cysteine decarboxylases including the lantibiotic-biosynthesizing enzymes EpiD and MrsD, all of which use a tightly bound flavin cofactor to oxidize the thiol moiety of the substrate to a thioaldehyde. The thioaldehyde serves to delocalize the charge that develops in the subsequent decarboxylation reaction. In the case of PPC-DC enzymes the resulting enethiol is reduced to a thiol giving net decarboxylation of cysteine, while in EpiD and MrsD it is released as the final product of the reaction. In this paper, we describe the characterization of the novel cyclopropyl-substituted product analogue 4'-phospho-N-(1-mercaptomethyl-cyclopropyl)-pantothenamide (PPanDeltaSH) as a mechanism-based inhibitor of the human PPC-DC enzyme. This inhibitor alkylates the enzyme on Cys(173), resulting in the trapping of a covalently bound enethiolate intermediate. When Cys(173) is exchanged for the weaker acid serine by site-directed mutagenesis the enethiolate reaction intermediate also accumulates. This suggests that Cys(173) serves as an active site acid in the protonation of the enethiolate intermediate in PPC-DC enzymes. We propose that this protonation step is the key mechanistic difference between the oxidative decarboxylases EpiD and MrsD (which have either serine or threonine at the corresponding position in their active sites) and PPC-DC enzymes, which also reduce the intermediate in an overall simple decarboxylation reaction.