4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen 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-aminolevulinic 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 Commonwealth 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 Environmental 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 ...

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The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A-and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A-and P-sites is proposed. ß

Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active sitedirected irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGScatalyzed reaction.