Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity

4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible in hibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitam in Bit, 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 t ime-dependent and dramatic species-specific inhibition of PBGS enzymes. IC5 0 values vary from 1 muM to 2.4 mM for human, Escherichia coli, Bradyrhizob ium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizin g a catalytic Zn2+ are more sensitive to 4,7-DOSA than those that do not. W eak 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 form s a Schiff base intermediate to one substrate molecule. A 1.9 Angstrom reso lution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDE code 1I8J) shows one dimer per asymmetric unit and reveals that the inhibitor fo rms two Schiff base linkages with each monomer, one to the normal Schiff ba se-forming Lys-246 and the other to a universally conserved "perturbing" Ly s-194 (E. coli numbering). This is the first structure to show inhibitor bi nding at the second of two substrate-binding sites.