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Eileen K Jaffe - One of the best experts on this subject based on the ideXlab platform.
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Porphobilinogen Synthase an equilibrium of different assemblies in human health
Progress in Molecular Biology and Translational Science, 2020Co-Authors: Eileen K JaffeAbstract:Abstract Porphobilinogen Synthase (PBGS) is an essential enzyme that catalyzes an early step in heme biosynthesis. An unexpected human PBGS quaternary structure dynamic drove the definition of morpheeins, which are protein multimers that dissociate, change shape, and re-assemble differently with functional consequences. Each PBGS monomer has two domains that can reposition through a hinge motion. Human PBGS exists in an equilibrium among high activity octamer, low activity hexamer, and low mole-fraction dimer in which the hinge motion occurs. The dimer conformation dictates the multimer architecture. An octamer-specific inter-subunit interaction responds to pH, resulting in a pH-dependence to the octamer-hexamer equilibrium. An inborn error of metabolism, ALAD porphyria, is caused by single amino acid substitutions that stabilize the hexamer relative to octamer. Drugs that stabilize the PBGS hexamer result in a drug side effect that can exacerbate porphyria. PBGS is essential for all organisms that require respiration, photosynthesis, or methanogenesis. Consequently, phylogenetic variation in PBGS multimerization equilibria provides insight into how Nature has harnessed oligomeric variation in the control of protein function. The dynamic multimerization of PBGS revealed the morpheein mechanism for allostery, a structural basis for inborn errors of metabolism, a quaternary structure focus for drug discovery and/or drug side effects, and a pathway toward new antibiotics or herbicides. The fortuitous discovery of PBGS quaternary structure dynamics arose from characterization of a low-activity single amino acid variant that dramatically stabilized the hexamer, whose existence had previously gone unnoticed.
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the remarkable character of Porphobilinogen Synthase
Accounts of Chemical Research, 2016Co-Authors: Eileen K JaffeAbstract:ConspectusPorphobilinogen Synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn2+; a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb2+. PBGSs that do not require the soft metal ion Zn2+ at the active site instead are suspected of using the hard metal Mg2+.The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid m...
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waladin benzimidazoles differentially modulate the function of Porphobilinogen Synthase orthologs
Journal of Medicinal Chemistry, 2014Co-Authors: Christian S Lentz, Eileen K Jaffe, Victoria S Halls, Jeffrey S Hannam, Silke Strassel, Sarah H Lawrence, Michael Famulok, Achim Hoerauf, Kenneth PfarrAbstract:The heme biosynthesis enzyme Porphobilinogen Synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg2+, or K+ stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design...
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Allostery and the dynamic oligomerization of Porphobilinogen Synthase.
Archives of Biochemistry and Biophysics, 2011Co-Authors: Eileen K Jaffe, Sarah H LawrenceAbstract:Porphobilinogen Synthase (PBGS) is an ancient metabolic enzyme responsible for the biosynthesis of the fundamental monopyrrole that is the building block for myriad tetrapyrrolic cofactors required for life (porphyrin, chlorophyll, vitamin B12, siroheme, phytobilin, cofactor F430, etc.) [1]. The PBGS catalyzed reaction is an asymmetric condensation of two molecules of the substrate 5-aminolevulinic acid (ALA). All organisms that carry out respiration, photosynthesis, or methanogenesis require PBGS, and the enzyme is remarkably conserved throughout evolution [2]. The metabolic pathway from Porphobilinogen to the tetrapyrroles is phylogenetically variable but universally populated by photoreactive intermediates whose accumulation can be toxic [3–4]. Thus, as part of the control of tetrapyrrole biosynthesis, PBGS evolved an allosteric regulation mechanism. Unexpectedly, PBGS allostery involves alternate, functionally distinct multimeric assemblies whose architectures are so different as to require multimer dissociation as part of the interconversion between active and inactive assemblies [5]. Novel aspects of the allosteric regulation of PBGS led us to define a morpheein model of allostery (described elsewhere [6]); proteins that use this allosteric mechanism display properties that are characteristically different from proteins that follow the classic Monod-Wyman-Changeux and Koshland-Nemethy-Filmer models [7]. Herein, we describe the phylogenetically variable allosteric regulation of PBGS; presumably these variations evolved due to the considerably different environments that organisms inhabit and the phylogenetic variation in the subcellular location of PBGS. In most species PBGS is located in the cytosol [8–9]. However, in plants it is in the chloroplast [10], and in apicomplexan parasites it is in the apicoplast [9].
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crystal structure of toxoplasma gondii Porphobilinogen Synthase insights on octameric structure and Porphobilinogen formation
Journal of Biological Chemistry, 2011Co-Authors: Eileen K Jaffe, Dhanasekaran Shanmugam, Anna S Gardberg, Shellie H Dieterich, Banumathi Sankaran, Lance Stewart, Peter J Myler, David S RoosAbstract:Porphobilinogen Synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product Porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.
Marina Volin - One of the best experts on this subject based on the ideXlab platform.
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mechanistic implications of mutations to the active site lysine of Porphobilinogen Synthase
Journal of Biological Chemistry, 2001Co-Authors: Laura W Mitchell, Jacob Martins, Marina Volin, Eileen K JaffeAbstract:Abstract 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.13C NMR studies of [3,5-13C]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. coliPBGS 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.
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Porphobilinogen Synthase from pea expression from an artificial gene kinetic characterization and novel implications for subunit interactions
Biochemistry, 2000Co-Authors: Jukka Kervinen, Samuel Litwin, Jacob Martins, Roland L Dunbrack, Marina Volin, Robert C Scarrow, Anthony T Yeung, And Erica Yoon, Eileen K JaffeAbstract:Porphobilinogen Synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B12. The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor Porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pKa values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kine...
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an artificial gene for human Porphobilinogen Synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning
Journal of Biological Chemistry, 2000Co-Authors: Eileen K Jaffe, Jacob Martins, Roland L Dunbrack, Marina Volin, Jukka Kervinen, Colleen R Bronsonmullins, Jack F Quinlan, Matthew H Sazinsky, Erica M Steinhouse, Anthony T YeungAbstract:Abstract Porphobilinogen Synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B12). 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.
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crystallization and preliminary x ray diffraction studies of e coli Porphobilinogen Synthase and its heavy atom derivatives
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: L Shimonilivny, Eileen K Jaffe, H L Carrell, T Wagner, Kaufman A Katz, Carol E Afshar, Laura W Mitchell, Marina Volin, Jenny P GluskerAbstract:Porphobilinogen Synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form Porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported. These crystals diffract to about 3.0 A resolution.
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crystallization and preliminary x ray diffraction studies of 5 chlorolevulinate modified bovine Porphobilinogen Synthase and the pbii complexed enzyme
Acta Crystallographica Section D-biological Crystallography, 1996Co-Authors: H L Carrell, Carol E Afshar, Marina Volin, Jenny P Glusker, Liat Shimoni, L J Keefe, Eileen K JaffeAbstract:: Bovine Porphobilinogen Synthase (PBGS) is an homo-octameric enzyme with four active sites. Each active site binds two Zn(II) atoms whose ligands differ and two molecules of 5-aminolevulinate whose chemical fates differ. The asymmetric binding of two Zn(II) atoms and two identical substrate molecules by a homodimeric active site is apparently unique. Modification by 5-chiorolevulinate can be used to differentiate the two substrate-binding sites; diffraction-quality crystals of 5-chlorolevulinate-modified PBGS have been obtained. Pb(II) can be used to differentiate the two different Zn(II)-binding sites; diffraction-quality crystals of the Pb(II) complex of PBGS have been obtained. Preliminary diffraction data reveal an I422 space group, in agreement with a general model for the quaternary structure of PBGS.
Leif A Eriksson - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of Porphobilinogen Synthase the chemical step revisited by qm mm calculations
Journal of Physical Chemistry B, 2012Co-Authors: Boxue Tian, Edvin Erdtman, Leif A ErikssonAbstract:Porphobilinogen Synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give Porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C–C bond formation first mechanism and the C–N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism.
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catalytic mechanism of Porphobilinogen Synthase the chemical step revisited by qm mm calculations b
The Journal of Physical Chemistry, 2012Co-Authors: Boxue Tian, Edvin Erdtman, Leif A ErikssonAbstract:Porphobilinogen Synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give Porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C–C bond formation first mechanism and the C–N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism.
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computational insights into the mechanism of Porphobilinogen Synthase
Journal of Physical Chemistry B, 2010Co-Authors: Edvin Erdtman, Eric A C Bushnell, James W Gauld, Leif A ErikssonAbstract:Porphobilinogen Synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of Porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersub ...
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modelling the mechanism of Porphobilinogen Synthase
2010Co-Authors: Edvin Erdtman, James W Gauld, Leif A ErikssonAbstract:5-aminolevulinic acid (5-ALA) and derivatives thereof are widely usedprodrugs in treatment of pre-malignant skin diseases of the cancer treatmentmethod photodynamic therapy (PDT). The target molecule in 5-ALAPDTis protoporphyrin IX (PpIX), which is synthesized endogenously from5-ALA via the heme pathway in the cell. This thesis is focused on 5-ALA,which is studied in different perspectives and with a variety of computationalmethods. The structural and energetic properties of 5-ALA, itsmethyl-, ethyl- and hexyl esters, four different 5-ALA enols, and hydrated5-ALA have been investigated using Quantum Mechanical (QM) first principlesdensity functional theory (DFT) calculations. 5-ALA is found to bemore stable than its isomers and the hydrolysations of the esters are morespontaneous for longer 5-ALA ester chains than shorter. The keto-enoltautomerization mechanism of 5-ALA has been studied, and a self-catalysismechanism has been proposed to be the most probable. Molecular Dynamics(MD) simulations of a lipid bilayer have been performed to study themembrane permeability of 5-ALA and its esters. The methyl ester of 5-ALAwas found to have the highest permeability constant (PMe-5-ALA = 52.8 cm/s).The mechanism of the two heme pathway enzymes; Porphobilinogen Synthase(PBGS) and Uroporphyrinogen III decarboxylase (UROD), have beenstudied by DFT calculations and QM/MM methodology. The rate-limitingstep is found to have a barrier of 19.4 kcal/mol for PBGS and 13.7kcal/mol for the first decarboxylation step in UROD. Generally, the resultsare in good agreement with experimental results available to date.
Reinhard Neier - One of the best experts on this subject based on the ideXlab platform.
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synthesis of bisubstrate inhibitors of Porphobilinogen Synthase from pseudomonas aeruginosa
Chemistry & Biodiversity, 2007Co-Authors: S Gacond, F Frere, M Nentwich, Nicole Frankenbergdinkel, Jeanphilippe Faurite, Reinhard NeierAbstract:: Porphobilinogen Synthase (PBGS) synthesizes Porphobilinogen 2 (PBG), the common precursor of all natural tetrapyrroles, through an asymmetric condensation of two molecules of 5-aminolevulinic acid 1 (ALA). Symmetrically linked dimers 7-11 derived from levulinic acid 3 (gamma-oxovaleric acid) have been synthesized to mimic the assumed bisubstrate bound to the active site of the enzyme. Their inhibition potential was characterized by determination of the IC(50) and K(i) values using PBGS from Pseudomonas aeruginosa. The polarity and the size of the functional group linking the two levulinic acid 3 units have a strong influence on the inhibition behavior.
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probing the active site of pseudomonas aeruginosa Porphobilinogen Synthase using newly developed inhibitors
Biochemistry, 2006Co-Authors: F Frere, Dirk W Heinz, M Nentwich, S Gacond, Reinhard Neier, Nicole FrankenbergdinkelAbstract:: Porphobilinogen Synthase catalyzes the first committed step of the tetrapyrrole biosynthesis pathway. In an aldol-like condensation, two molecules of 5-aminolevulinic acid (ALA) form the first pyrrole, Porphobilinogen. Newly synthesized analogues of a reaction intermediate of Porphobilinogen Synthase have been employed in studying the active site and the catalytic mechanism of this early enzyme of tetrapyrrole biosynthesis. This study combines structural and kinetic evaluation of the inhibition potency of these inhibitors. In addition, one of the determined protein structures provides for the first time structural evidence of a magnesium ion in the active site. From these results, we can corroborate an earlier postulated enzymatic mechanism that starts with formation of a C-C bond, linking C3 of the A-side ALA to C4 of the P-side ALA through an aldole addition. The obtained data are discussed with respect to the current literature.
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structure of Porphobilinogen Synthase from pseudomonas aeruginosa in complex with 5 fluorolevulinic acid suggests a double schiff base mechanism
Journal of Molecular Biology, 2002Co-Authors: F Frere, Nicole Frankenberg, Dieter Jahn, Wolfdieter Schubert, Reinhard Neier, Frederic Stauffer, Dirk W HeinzAbstract:Abstract All natural tetrapyrroles, including hemes, chlorophylls and vitamin B 12 , share Porphobilinogen (PBG) as a common precursor. Porphobilinogen Synthase (PBGS) synthesizes PBG through the asymmetric condensation of two molecules of aminolevulinic acid (ALA). Crystal structures of PBGS from various sources confirm the presence of two distinct binding sites for each ALA molecule, termed A and P. We have solved the structure of the active-site variant D139N of the Mg 2+ -dependent PBGS from Pseudomonas aeruginosa in complex with the inhibitor 5-fluorolevulinic acid at high resolution. Uniquely, full occupancy of both substrate binding sites each by a single substrate-like molecule was observed. Both inhibitor molecules are covalently bound to two conserved, active-site lysine residues, Lys205 and Lys260, through Schiff bases. The active site now also contains a monovalent cation that may critically enhance enzymatic activity. Based on these structural data, we postulate a catalytic mechanism for P. aeruginosa PBGS initiated by a C–C bond formation between A and P-side ALA, followed by the formation of the intersubstrate Schiff base yielding the product PBG.
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species specific inhibition of Porphobilinogen Synthase by 4 oxosebacic acid
Journal of Biological Chemistry, 2002Co-Authors: Eileen K Jaffe, Reinhard Neier, Jacob Martins, Jukka Kervinen, Alexander Wlodawer, Frederic Stauffer, Alexander ZdanovAbstract: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.
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inhibition studies of Porphobilinogen Synthase from escherichia coli differentiating between the two recognition sites
ChemBioChem, 2001Co-Authors: Frederic Stauffer, Jeanphilippe Faurite, Eleonora Zizzari, Caroline Engelochjarret, Janette Bobalova, Reinhard NeierAbstract:substrates ending up in the A site or in the P site of Porphobilinogen Synthase. Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites. The P site, whose structure is known from X-ray crystallographic studies, tolerates ester functions well. The A site interacts very strongly with nitro groups, but is not very tolerant to ester functions. This differentiation is a central factor in the asymmetric handling of the two identical substrates. Finally, it could be shown that the keto group of the substrate bound at the A site is not only essential for the recognition, but that an increase in electrophilicity of the carbon atom also increases the inhibition potency considerably. This has important consequences for the recognition process at the A site, whose exact structure is not yet known.
Laura W Mitchell - One of the best experts on this subject based on the ideXlab platform.
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mechanistic implications of mutations to the active site lysine of Porphobilinogen Synthase
Journal of Biological Chemistry, 2001Co-Authors: Laura W Mitchell, Jacob Martins, Marina Volin, Eileen K JaffeAbstract:Abstract 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.13C NMR studies of [3,5-13C]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. coliPBGS 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.
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crystallization and preliminary x ray diffraction studies of e coli Porphobilinogen Synthase and its heavy atom derivatives
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: L Shimonilivny, Eileen K Jaffe, H L Carrell, T Wagner, Kaufman A Katz, Carol E Afshar, Laura W Mitchell, Marina Volin, Jenny P GluskerAbstract:Porphobilinogen Synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form Porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported. These crystals diffract to about 3.0 A resolution.
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the phylogenetically conserved histidines of escherichia coli Porphobilinogen Synthase are not required for catalysis
Journal of Biological Chemistry, 1995Co-Authors: Laura W Mitchell, Marina Volin, Eileen K JaffeAbstract:Abstract Porphobilinogen Synthase (PBGS) is a metalloenzyme that catalyzes the first common step of tetrapyrrole biosynthesis, the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form Porphobilinogen. Chemical modification data implicate histidine as a catalytic residue of PBGS from both plants and mammals. Histidine may participate in the abstraction of two non-ionizable protons from each substrate molecule at the active site. Only one histidine is species-invariant among 17 known sequences of PBGS which have high overall sequence similarity. In Escherichia coli PBGS, this histidine is His128. We performed site-directed mutagenesis on His128, replacing it with alanine. The mutant protein H128A is catalytically active. His128 is part of a histidine- and cysteine-rich region of the sequence that is implicated in metal binding. The apparent Kd for Zn(II) binding to H128A is about an order of magnitude higher than for the wild type protein. E. coli PBGS also contains His126 which is conserved through the mammalian, fungal, and some bacterial PBGS. We mutated His126 to alanine, and both His126 and His128 simultaneously to alanine. All mutant proteins are catalytically competent; the Vmax values for H128A (44 units/mg), H126A (75 units/mg), and H126/128A (61 units/mg) were similar to wild type PBGS (50 units/mg) in the presence of saturating concentrations of metal ions. The apparent Kd for Zn(II) of H126A and H126/128A is not appreciably different from wild type. The activity of wild type and mutant proteins are all stimulated by an allosteric Mg(II); the mutant proteins all have a reduced affinity for Mg(II). We observe a pKa of ∼7.5 in the wild type PBGS kcat/Km pH profile as well as in those of H128A and H126/128A, suggesting that this pKa is not the result of protonation/deprotonation of one of these histidines. H128A and H126/128A have a significantly increased Km value for the substrate ALA. This is consistent with a role for one or both of these histidines as a ligand to the required Zn(II) of E. coli PBGS, which is known to participate in substrate binding. Past chemical modification may have inactivated the PBGS by blocking Zn(II) and ALA binding. In addition, the decreased Km for E. coli PBGS at basic pH allows for the quantitation of active sites at four per octamer.
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characterization of the role of the stimulatory magnesium of escherichia coli Porphobilinogen Synthase
Biochemistry, 1995Co-Authors: Eileen K Jaffe, Laura W Mitchell, Marina Volin, Kathleen M Taylor, George D MarkhamAbstract:: The synthesis of tetrapyrroles is essential to all phyla. Porphobilinogen Synthase (PBGS) is a zinc metalloenzyme that catalyzes the formation of Porphobilinogen, the monopyrrole precursor of all biological tetrapyrroles. The enzyme from various organisms shows considerable sequence conservation, suggesting a common fold, quaternary structure, and catalytic mechanism. Escherichia coli and plant PBGS are activated by magnesium, a property that is absent from mammalian PBGS. This stimulatory Mg(II) is called Mgc. Mgc is not required for activity and is distinct from the two zinc ions (ZnA and ZnB) common to mammalian and E. coli PBGS (PBGSE.coli). For PBGSE.coli, both the Km for the substrate 5-aminolevulinic acid (ALA) and the Vmax are altered by the presence of Mgc; Mg(II) causes the Km to drop from approximately 3 to 0.30 mM and the maximum specific activity to increase from 23 to 50 mumol h-1 mg-1. Mgc also causes the saturating concentration of the required Zn(II) to decrease from 0.1 mM to 10 microM. Maximal activation by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mgc site is probably saturated under physiological conditions. Mn(II) is a good substitute for Mgc, giving a comparable increase in catalytic activity. Consequently, Mn(II) has been used as an EPR active probe of the Mgc binding site. Mn(II) binds at a stoichiometry of eight ions per enzyme octamer. The X- and Q-band EPR spectra reflect a single type of binding site with rhombic symmetry and are consistent with oxygen and/or nitrogen ligands. The addition of unlabeled or 1-13C-labeled ALA does not significantly affect the Mn(II) EPR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)
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Porphobilinogen Synthase from escherichia coli is a zn ii metalloenzyme stimulated by mg ii
Archives of Biochemistry and Biophysics, 1993Co-Authors: Laura W Mitchell, Eileen K JaffeAbstract:Abstract Porphobilinogen Synthase (PBGS) is essential to all life forms; in mammals it is definitively established that Zn(II) is required for activity. The literature regarding the metal requirement for PBGS in other animals, plants, and bacteria neither establishes nor disproves a Zn(II) requirement. We have characterized Escherichia coli PBGS and found it to be remarkably similar to bovine PBGS. The similarities include a requirement for Zn(II), inhibition by 1,10-phenanthroline, an exceptional thermal stability, a requirement for free sulfhydryl(s) as shown by sensitivity to modification by methyl methanethiosulfonate, and the presence of tightly bound product on freshly isolated enzyme. Proton-induced X-ray emission analysis shows E. coli PBGS to contain a stoichiometric amount of Zn and no other metals. The most striking similarity between E. coli and bovine PBGS is the 13 C NMR spectrum of enzyme-bound [3,5- 13 C]PBG; the chemical shifts of bound product are identical for both bovine and E. coli PBGS. Minor differences between E. coli PBGS and its mammalian counterpart include K m (ALA) = 1.9 mM, a pH optimum of 7.5-8, and its molar absorbtion coefficient expressed as A 0.1% 280 is 0.588. We conclude from these data that E. coli PBGS is a Zn(II)-metalloenzyme and that Zn(II) is required for catalytic activity, and propose that the mammalian and bacterial PBGS function by similar mechanisms. There is one significant difference between E. coli and mammalian PBGS. For E. coli PeGS, Mg(II) causes a twofold stimulation of the Zn(II)-induced E. coli PBGS activity; this effect is not seen for bovine PBGS. The stimulation of activity by Mg(II) mimics the effect of Mg(II) on plant PBGS, although E. coli PBGS does not contain the putative Mg(II) binding site recently revealed by Boese et al. [Q. F. Boese, A. J. Spano, T. Li, and M. P. Timko (1991) J. Biol. Chem. 266, 17060-170661. This work lays the foundation for identification of functional amino acids based on the sequence similarities between PBGS from bacterial, plant, and mammalian sources.