The molecular mechanism of lead inhibition of human porphobilinogen synthase

Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles of Zn(II) and Pb(II), we produced human PBGS mutants that are designed to lack either the ZnA or ZnB sites. These proteins, MinusZnA (H131A, C223A) and MinusZnB (C122A, C124A, C132A), each become purified with four Zn(II) per octamer, thus confirming an asymmetry in the human PBGS structure. MinusZnA is fully active, whereas MinusZnB is far less active, verifying an important catalytic role for ZnB and the removed cysteine residues. Kinetic properties of the mutants and wild type proteins are described. Comparison of Pb(II) inhibition of the mutants shows that ligands to both ZnA and ZnB interact with Pb(II). The ZnB ligands preferentially interact with Pb(II). At least one ZnA ligand is responsible for the slow tight binding behavior of Pb(II). The data support a novel model where a high affinity lead site is a hybrid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb(II) precludes Pb(II) to function in PBGS catalysis.     

Allosteric Inhibition of Human Porphobilinogen Synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.     

Synthesis and antibacterial activity of alaremycin derivatives for the porphobilinogen synthase

The preparation and the antibacterial activity of alaremycin derivatives such as their CF₃-derivatives and (R)- and (S)-4-oxo-5-acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described. The IC₅₀ values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were determined using PBGS assay.     

An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.     

Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.

During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST). After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme. Mg2+ stabilized the oligomeric state but was not essential for octamer formation. Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity. EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+. At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme. Pb2+, Cd2+, and Hg2+ did not restore activity. A stimulatory effect of monovalent ions was observed. A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found. pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2). These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis. Some PBGS inhibitors were characterized. Finally, we succeeded in obtaining well-ordered crystals of P. aeruginosa PBGS complexed with the substrate analogue levulinic acid.     

Investigations on the metal switch region of human porphobilinogen synthase

Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis. The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium. In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium. The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS. The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc. The specific activities of the chimeras are significantly lower than human PBGS. Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage. The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence. Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position. The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.     

Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

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 (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-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 ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-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.     

Porphobilinogen synthase,the first source of Heme's asymmetry

Porphobilinogen is the monopyrrole precursor of all biological tetrapyrroles. The biosynthesis of porphobilinogen involves the asymmetric condensation of two molecules of 5-aminolevulinate and is carried out by the enzyme porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase. This review documents what is known about the mechanism of the PBGS-catalyzed reaction. The metal ion constitutents of PBGS are of particular interest because PBGS is a primary target for the environmental toxin lead. Mammalian PBGS contains two zinc ions at each active site. Bacterial and plant PBGS use a third metal ion, magnesium, as an allosteric activator. In addition, some bacterial and plant PBGS may use magnesium in place of one or both of the zinc ions of mammalian PBGS. These phylogenetic variations in metal ion usage are described along with a proposed rationale for the evolutionary divergence in metal ion usage. Finally, I describe what is known about the structure of PBGS, an enzyme which has as yet eluded crystal structure determination.     

Tracking the evolution of porphobilinogen synthase metal dependence in vitro

Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.     

Developmental profiles of 5-aminolevulinate, porphobilinogen and porphobilinogen synthase activity in Pieris brassicae related to the synthesis of the bilin-binding protein

The bilin-binding protein (BBP) occurs as a major soluble protein in haemolymph, fat body, epidermis and wings of Pieris brassicae. It is a member of the lipocalin protein superfamily with yet unknown function. Here, we studied the developmental regulation of tetrapyrrole biosynthesis that provides the bilin ligand as the predominating end product. The levels of the precursors 5-aminolevulinate (ALA) and porphobilinogen (PBG) varied during larval-pupal transition in accordance with the activity of the related enzyme porphobilinogen synthase (PBGS). During adult development, both precursors were low while PBGS activity increased parallel to the formation of BBP, as shown in previous work. A competitive inhibitor of PBGS was partially purified from the meconium and characterised as a heat-stabile acidic compound. Label from [14C]ALA, injected into developing pupae of different age, was found to 80% in the hind wings and to 20% in the forewings after adult eclosion, reflecting the unequal distribution of BBP between the pairs of wings. This contrasted to the activity of PBGS that was equally active in forewings and hind wings. Together with the variation of enzyme activity during wing development our results led us propose that the (hind) wings may play a role in the synthesis of the tetrapyrrole ligand of BBP.     

A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.     

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.     

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.     

ALAD porphyria is a conformational disease

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.     

Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid

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 site-directed 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 PBGS-catalyzed reaction.     

Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. (13)C NMR studies of [3,5-(13)C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.     

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins)

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.     

Substrate-induced interconversion of protein quaternary structure isoforms

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. 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.     

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys²²³ is transferred in the active center to be coordinated by Cys¹²², Cys¹²⁴, and Cys¹³² (Sawada et al. in J Biol Inorg Chem 10:199–207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f ₀. It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys¹²², Cys¹²⁴, and Cys¹³² and coordination with zinc ion is closely linked to change in the oligomeric state.