Mechanistic Basis for Suicide Inactivation of Porphobilinogen Synthase by 4,7-Dioxosebacic Acid, an Inhibitor That Shows Dramatic Species Selectivity † Jukka Kervinen, ‡ Eileen K. Jaffe,* ,‡ Fre ´de ´ric Stauffer, § Reinhard Neier, § Alexander Wlodawer, | and Alexander Zdanov | Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme AVenue, Philadelphia, PennsylVania 19111, Department of Chemistry, UniVersity of Neuchatel, Neuchatel 2007, Switzerland, and Macromolecular Structure Laboratory, National Cancer Institute, Frederick, Maryland 21702 ReceiVed April 2, 2001; ReVised Manuscript ReceiVed May 15, 2001 ABSTRACT: 4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porpho- bilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin B 12 , and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-amino- levulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC 50 values vary from 1 µM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn 2+ are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 Å resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code 1I8J) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved “perturbing” Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites. Porphobilinogen synthase (PBGS, 1 EC 4.2.1.24, also known as 5-aminolevulinic acid dehydratase) is a highly conserved metalloenzyme that functions in the first common step of the biosynthesis of the essential tetrapyrroles. The PBGS-catalyzed reaction is an asymmetric condensation between two molecules of 5-aminolevulinic acid (ALA) as described in Figure 1A. The crystal structure of the enzyme has been established for PBGS from yeast, Escherichia coli, and Pseudomonas aeruginosa (1-3). Some of the published structures contain the bound inhibitor levulinic acid, a complex analogous to the first enzyme-bound intermediate (2-4), which is a Schiff base formed between the keto group of P-side ALA (see Figure 1A) and the amino group of an invariant lysine residue (see Figure 2A). These structures delineated the locations of several different divalent metal ions and the binding residues for the carboxylic acid moieties of the two ALA substrate molecules. Despite the fact that PBGS is an octamer of ∼300 kDa, the catalytic and binding residues at each active site derive from only one subunit. † This work was supported by Grant ES03654 (E.K.J.) from the National Institute of Environmental Health Sciences, NIH, by NIH Grant CA06927 (ICR), and by an appropriation from the Com- monwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute nor of the National Institute of Environ- mental Health Sciences. * To whom correspondence and reprint requests should be addressed. Telephone: 215-728-3695. Fax: 215-728-2412. E-mail: EK_Jaffe@fccc.edu. ‡ Fox Chase Cancer Center. § University of Neuchatel. | National Cancer Institute. 1 Abbreviations: PBGS, porphobilinogen synthase; ALA, 5-amino- levulinic acid; ME, 2-mercaptoethanol; BTP, 1,3-bis[tris(hydroxy- methyl)methylamino]propane; 4,7-DOSA, 4,7-dioxosebacic acid. FIGURE 1: PBGS-catalyzed reaction and active site-directed inhibi- tors. (A) PBGS catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). A-side ALA (normal font) contributes the acetyl side chain and retains a free amino group. P-side ALA (bold bonds, italic font) contributes the propionyl side chain and has its amino group incorporated into the pyrrole ring. (B) PBGS inhibitors levulinic acid, 4,7-DOSA, and succinyl acetone. 10.1021/bi010656k CCC: $20.00 © xxxx American Chemical Society PAGE EST: 9.9 Published on Web 00/00/0000