Mechanistic Basis for Suicide Inactivation of Porphobilinogen Synthase by 4,7-Dioxosebacic Acid, an Inhibitor That Shows Dramatic Species Selectivity

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106

(2003) Nat. Struct. Biol. 10, 757-763). The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.A recent description of a rare human allele of porphobilinogen synthase (PBGS) 1 revealed that the enzyme can exist as two alternate quaternary structures wherein the oligomeric state is dictated by distinctly different structures for their component crystallographic asymmetric unit (1). The monomer is an ␣␤-barrel protein with a 23-residue N-terminal arm. The octameric form is comprised of four "hugging dimers" (Fig. 1a), whereas the hexameric form is comprised of three "detached dimers" (Fig. 1b). In both oligomeric assemblies there are invariant barrel-to-barrel subunit interactions within the dimers and quasiequivalent arm-to-bottom-of-barrel subunit interactions between adjacent dimers (Fig. 1). However, the octamer has a unique "hugging" arm-to-barrel interaction that is not seen in the hexamer.Preparations of heterologously expressed wild type human PBGS predominantly contain octamer, but there is a small propensity to adopt the hexameric structure (1). In contrast, heterologous expression of the rare human variant, F12L, yields protein that purifies as the hexamer and does not readily take on the octameric structure. The homo-octameric wild type and homohexameric F12L forms of human PBGS exhibit dramatic differences in pH activity profile and in the kinetic K m values (Table I) (1) despite the fact that amino acid residue 12 does not interact directly with active site residues in either quaternary structure isoform (Fig. 1). However, the arm-tobarrel interface found in the hugging dimer provides substantial stabilization for the solvent-accessible face of the ␣␤-barrel wherein lies the active site. This region of the protein, which is disordered in the hexamer, includes an ϳ11-amino acid stretch that normally serves to gate the active site from solvent, also called the active site lid. With the active site more solventaccessible in the hexamer, a high external pH is required to drive the Schiff base formation that is necessary for catalysis (2); this contributes to the high pH required for F12L activity. The decreased affinity of F12L for substrate is attributed to the loss of interactions between the destabilized active site lid and the carboxyl moiety of the outermost substrate (3).Coexpression of wild type and F12L (WTϩF12L...

Section: Discussionmentioning

confidence: 91%

Section: Dynamic Interconversion Of Wtϩf12l Hetero-oligomeric Pbgs Qumentioning

confidence: 99%

mentioning

confidence: 99%

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Substrate-induced Interconversion of Protein Quaternary Structure Isoforms

Tang

Stith

Jaffe

2005

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106

“…4A, the dose-response curve for succinylacetone inhibition of TgPBGS is comparable with that of HsPBGS, with IC 50 values of ϳ1 M. This is to be expected because the inhibitor binds covalently to a conserved active site lysine, causing suicide inhibition (39). In contrast, the active site-directed, species-specific irreversible inhibitor 4,7-dioxosebacic acid, which is most effective against Zn 2ϩ -requiring PBGS (28,37,40), shows ϳ40-fold selectivity for HsPBGS relative to TgPBGS (Fig. 4B).…”

Section: Inhibition Of Tgpbgs and Heme Biosynthesis In T Gondii-mentioning

confidence: 99%

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii

Shanmugam

Ramirez

et al. 2010

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Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/ fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn 2؉ and Mg 2؉ ) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ϳ320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7-8.5), with a K m for aminolevulinic acid of ϳ150 M and specific activity of ϳ24 mol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg 2؉ but not Zn 2؉ and shows two Mg 2؉ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg 2؉ -binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.

“…1A). Despite the fact that much is known about the PBGS structure, the sequence of bond-making and bond-breaking events that follow formation of the ternary complex of PBGS with the two substrate molecules is the subject of active discussion (2)(3)(4)(5). Based on an extensive phylogenetic variation in the number and kinds of metal ions used for catalytic and/or allosteric roles, it is possible that the order of chemical events in the enzymecatalyzed reaction mechanism may not be phylogenetically conserved.…”

mentioning

confidence: 99%

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic Acid

Jaffe¹,

Kervinen²,

Martins³

et al. 2002

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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.