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 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 microM 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 A resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code ) 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.
Kervinen
J
J
Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA.
Jaffe
E K
EK
Stauffer
F
F
Neier
R
R
Wlodawer
A
A
Zdanov
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D003652:Decanoic Acids / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D012545:Schiff Bases / Q000737:chemistry; D013045:Species Specificity; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry
C434703:4,7-dioxosebacic acid; D002413:Cations, Divalent; D003652:Decanoic Acids; D004791:Enzyme Inhibitors; D012545:Schiff Bases; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc