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A Structural Basis for Half-of-the-sites Metal Binding Revealed in Drosophila melanogaster Porphobilinogen Synthase

2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês

10.1074/jbc.m304124200

1083-351X

Lenka Kundrat, Jacob Martins, Linda Stith, Roland L. Dunbrack, Eileen K. Jaffe,

Folate and B Vitamins Research

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 K d well below μm. A second inhibitory zinc exhibits a K d of ∼5 μm 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. 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 K d well below μm. A second inhibitory zinc exhibits a K d of ∼5 μm 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. The porphobilinogen synthases (PBGS) 1The abbreviations used are: PBGS, porphobilinogen synthase; 4-OSA, 4-oxosebacic acid; 4,7-DOSA, 4,7-dioxosebacic acid; DmPBGS, D. melanogaster porphobilinogen synthase; β-ME, 2-mercaptoethanol; ALA, 5-aminolevulinic acid; o-phe, 1,10-phenanthroline.1The abbreviations used are: PBGS, porphobilinogen synthase; 4-OSA, 4-oxosebacic acid; 4,7-DOSA, 4,7-dioxosebacic acid; DmPBGS, D. melanogaster porphobilinogen synthase; β-ME, 2-mercaptoethanol; ALA, 5-aminolevulinic acid; o-phe, 1,10-phenanthroline. are a family of highly homologous homo-octameric proteins responsible for catalyzing the first common step in the biosynthesis of a broad range of tetrapyrrole pigments such as heme, vitamin B12, chlorophyll, and cofactor F430 of the methanogenic bacteria (1Shoolingin-Jordan P.M. Biochem. Soc. Trans. 1998; 26: 326-336Crossref PubMed Scopus (18) Google Scholar). PBGS is also known as 5-aminolevulinate dehydratase or ALAD. The most significant phylogenetic difference among PBGS proteins is in the constellation of metal ions at catalytic and allosteric sites (2Jaffe E.K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 115-128Crossref PubMed Scopus (92) Google Scholar, 3Jaffe E.K. Chem. Biol. 2003; 10: 25-34Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Yeast and mammalian PBGS share the sequence determinants for two zinc binding sites, one of which is absent from the PBGS of any archaea, bacteria, protist, or photosynthetic eucarya (3Jaffe E.K. Chem. Biol. 2003; 10: 25-34Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The human and yeast PBGS both contain the sequence determinants for a catalytic active site zinc (also known as ZnB), as well as a second non-essential zinc (also known as ZnA) (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), but they differ in metal binding stoichiometry. For instance, in the case of human PBGS, the catalytic zinc shows half-of-the-sites binding at a stoichiometry of 4/homo-octamer (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) (PDB accession number 1E51), 2N. Miles-Davies, D. Thompson, J. B. Cooper, P. M. Shoolingin-Jordan, Protein Data Bank accession number 1E51.2N. Miles-Davies, D. Thompson, J. B. Cooper, P. M. Shoolingin-Jordan, Protein Data Bank accession number 1E51. whereas the fungal enzyme binds the catalytic zinc at 8/octamer (6Erskine P.T. Senior N. Awan S. Lambert R. Lewis G. Tickle I.J. Sarwar M. Spencer P. Thomas P. Warren M.J. Shoolingin-Jordan P.M. Wood S.P. Cooper J.B. Nat. Struct. Biol. 1997; 4: 1025-1031Crossref PubMed Scopus (131) Google Scholar) (PDB accession number 1AW5). Some bacterial PBGS show half-of-the-sites metal binding at catalytic and/or allosteric sites (e.g. Bradyrhizobium japonicum and Pseudomonas aeruginosa), whereas others (e.g. Escherichia coli) do not. The current sequence databases contain PBGS sequences from ∼130 different organisms. Of these, the PBGS of Drosophila melanogaster (DmPBGS) is the only complete non-mammalian and non-fungal PBGS sequence that shows the sequence determinants for the two zinc binding sites found in yeast and mammalian PBGS. The gene encoding PBGS is absent from the completed genome of Caenorhabditis elegans and not yet verified in other incomplete non-mammalian metazoan genomes such as that of Danio rerio. Hence, to help deduce the molecular determinants for expression of the half-of-the-sites metal binding phenomenon, we obtained the expressed sequence tag encoding DmPBGS and cloned, expressed, purified, and characterized the protein, as described herein. Unexpectedly, DmPBGS was found to interact with zinc somewhat differently from either mammalian or yeast PBGS. Investigation of these differences provides novel insight into the structural basis for the half-of-the-sites metal binding phenomenon. Characterization of DmPBGS is also significant because insects are among the most abundant metazoan species on earth and can act as agricultural pests or human disease vectors. For many insects, flight is essential and depends upon aerobic respiration; consequently, tetrapyrroles (hemes) play an important role. Hence, species-specific inhibition of tetrapyrrole biosynthesis might prove useful in control of insects and or insect-borne diseases. Because PBGS has recently been identified to have species-specific sensitivity to certain active site-directed inhibitors (7Kervinen J. Jaffe E.K. Stauffer F. Neier R. Wlodawer A. Zdanov A. Biochemistry. 2001; 40: 8227-8236Crossref PubMed Scopus (40) Google Scholar, 8Jaffe E.K. Kervinen J. Martins J. Stauffer F. Neier R. Wlodawer A. Zdanov A. J. Biol. Chem. 2002; 277: 19792-19799Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), we have characterized DmPBGS with these inhibitors. Cloning and Expression of DmPBGS—The expressed sequence tag containing the gene for DmPBGS (catalogue number 98002) was purchased from ResGen. The gene was amplified by PCR with the addition of the NdeI and BamHI sites, using the primers 5′-GCTAAGCGAACCATATGGAGCGGAAACTGC-3′ and 5′-CGCATGTACGGATCCACATGGTATCAAGACATCGG-3′, where the restriction sites are underlined. The amplified gene was digested with NdeI and BamHI and ligated directly into pET17b, which had also been digested with NdeI and BamHI. The sequences of several resulting plasmids were determined throughout the gene in both directions using a series of internal and external primers. An error-free plasmid was denoted pJMPBGS and was used for further study. pJMPBGS was transformed into E. coli strain BLR(DE3) and the CodonPlus variant BL21(DE3)-RIL for expression using the procedure we had previously used for pMVhum (9Jaffe E.K. Volin M. Bronson-Mullins C.R. Dunbrack Jr., R.L. Kervinen J. Martins J. Quinlan Jr., J.F. Sazinsky M.H. Steinhouse E.M. Yeung A.T. J. Biol. Chem. 2000; 275: 2619-2626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The DmPBGS mutant H10F was prepared by the QuikChange method (Stratagene) using the sense strand primer 5′-GCACAGTGGAATGTTCCATGCCACGCTGCGGC-3′. All oligonucleotides were synthesized in the Fannie E. Rippel Biochemistry and Biotechnology Facility at Fox Chase Cancer Center (FCCC). DNA sequence determination was in the FCCC DNA Sequencing Core Facility. DmPBGS purification initially followed the protocol for human PBGS expressed from pMVhum (9Jaffe E.K. Volin M. Bronson-Mullins C.R. Dunbrack Jr., R.L. Kervinen J. Martins J. Quinlan Jr., J.F. Sazinsky M.H. Steinhouse E.M. Yeung A.T. J. Biol. Chem. 2000; 275: 2619-2626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The protocol was altered as differences were observed. The final purification protocol followed the published procedures up to the ammonium sulfate cut, which was altered to use the protein that precipitated between 30 and 50% ammonium sulfate. This protein was redissolved in 30 mm KPi, pH 7.5, 10 μm Zn2+, 10 mm 2-mercaptoethanol (β-ME), 0.1 mm phenylmethylsulfonyl fluoride, and 10% ammonium sulfate, adsorbed to a 100-ml phenyl-Sepharose column, and eluted in 2 mm KPi, pH 7.5, 10 μm Zn2+, 10 mm β-ME, 0.1 mm phenylmethylsulfonyl fluoride following an 800-ml linear gradient to the final buffer. DmPBGS was pooled and applied directly to a 100-ml DEAE Biogel A column that was equilibrated in 2 mm KPi, pH 7.5, 10 μm Zn2+, 10 mm β-ME, 0.1 mm phenylmethylsulfonyl fluoride. The protein was eluted with a 1-liter gradient to 0.4 m KCl in 30 mm KPi, pH 7.5, 10 μm Zn2+, 10 mm β-ME, 0.1 mm phenylmethylsulfonyl fluoride. DmPBGS elutes in the first third of the gradient and is baseline separated from chromosomally encoded E. coli PBGS activity that elutes in the second half of the gradient. DmPBGS was pooled, concentrated to ∼10 mg/ml, and further purified on a 1-meter long 270-ml Sephacryl S300 column in 0.1 m KPi, pH 7.0, 10 μm Zn2+, 10 mm β-ME. Dry weight analysis was used to determine the extinction coefficient of DmPBGS as previously described (10Kervinen J. Dunbrack Jr., R.L. Litwin S. Martins J. Scarrow R.C. Volin M. Yeung A.T. Yoon E. Jaffe E.K. Biochemistry. 2000; 39: 9018-9029Crossref PubMed Scopus (37) Google Scholar). Building a Model of DmPBGS—Two models were prepared to approximate the structure of octameric DmPBGS. These were based on the crystal structures of yeast PBGS with aminolevulinic acid bound (PDB accession number 1H7P, 1.67 Å resolution; Ref. 11Erskine P.T. Newbold R. Brindley A.A. Wood S.P. Shoolingin-Jordan P.M. Warren M.J. Cooper J.B. J. Mol. Biol. 2001; 312: 133-141Crossref PubMed Scopus (39) Google Scholar) and human PBGS with the product porphobilinogen bound (PDB accession number 1E51, 2.83 Å resolution). The unit cell of the former is a monomer making a symmetric octamer, whereas the latter unit cell is an asymmetric dimer, thus making an asymmetric octamer; the models were originally built as monomer and dimer, respectively. Sequence alignments were performed with the BLAST program (12Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59401) Google Scholar) with some manual adjustment upon examination of the sequences in light of the structures. All insertions and deletions in the alignment occurred in coil regions located between regular secondary structure segments. For both models, loop modeling in insertion and deletion regions in the alignment was performed with the Modeler program (version 6) (13Fiser A. Do R.K. Sali A. Protein Sci. 2000; 9: 1753-1773Crossref PubMed Scopus (1613) Google Scholar), and side chain conformations were predicted with the program SCWRL (version 2.95) (14Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (482) Google Scholar, 15Dunbrack Jr., R.L. Cohen F.E. Protein Sci. 1997; 6: 1661-1681Crossref PubMed Scopus (667) Google Scholar, 16Dunbrack Jr., R.L. Proteins. 1999; 3: 81-87Crossref PubMed Scopus (66) Google Scholar). Side chains for residues that were identical in the DmPBGS sequence and the template proteins were kept fixed in their crystallographic Cartesian coordinates. The octamers were built using the symmetry transformations provided in the PDB entries with in-house custom software. The side chain calculations were repeated following formation of the octamer from the fundamental asymmetric units. Again, conserved amino acids were kept in their original positions. PBGS Activity Assays—Determinations of PBGS activity used a fixed-time assay at 37 °C in 0.1 m bis-tris propane-HCl, 10 mm β-ME, and the substrate 5-aminolevulinic acid (ALA), with variations in assay pH, concentrations of metal ions such as Zn2+, Mg2+, K+, and the Zn2+ chelator, 1,10-phenanthroline (o-phe). Procedures for pH versus activity profiles, K m and V max determinations, and inhibition by 4,7-dioxosebacic acid and 4-oxosebacic acid were as previously described (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Kervinen J. Jaffe E.K. Stauffer F. Neier R. Wlodawer A. Zdanov A. Biochemistry. 2001; 40: 8227-8236Crossref PubMed Scopus (40) Google Scholar). DmPBGS (10 μg/ml) activity was determined as a function of pH using a fixed assay time of 5 min, a fixed ALA concentration of 10 mm, with and without 10 μm Zn2+, 1 mm Mg2+, or 0.1 m K+. Reported pH values are final assay pH following addition of ALA-HCl. The o-phe inhibition studies were carried out such that each assay contained the appropriate amount of o-phe in 10 μl of ethanol (1% final volume). Equilibrium Dialysis Studies—Equilibrium dialysis was carried out at 37 ° for 4 h under gentle agitation in an air shaker. Protein solutions were ∼1 ml at ∼1 mg ml–1, and the dialysis buffer volume was 200 ml. Dialysis was carried out in 0.1 m bis-tris propane-HCl, 10 mm β-ME at pH 8 in the presence of 0.01–10 mm o-phe (±1 mm ALA) and in the presence of 0–30 μm Zn2+ (±1 mm ALA). Enzyme-bound zinc was determined by atomic absorption spectroscopy as previously described (9Jaffe E.K. Volin M. Bronson-Mullins C.R. Dunbrack Jr., R.L. Kervinen J. Martins J. Quinlan Jr., J.F. Sazinsky M.H. Steinhouse E.M. Yeung A.T. J. Biol. Chem. 2000; 275: 2619-2626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Protein Expression and Purification—The expression of DmPBGS from pJMPBGS was attempted in E. coli strains BLR(DE3) and BL21-CodonPlus-RIL. Based on SDS gels following a small scale growth, the CodonPlus host did not appear to greatly influence expression. However, because the native gene encoding DmPBGS contains clusters of rare codons, the CodonPlus strain was selected for expression in order to minimize the possibility of translational errors. From each 1-liter growth we obtained ∼10 g of cells, from which ∼45 mg of DmPBGS was purified to homogeneity. The final purification buffer was 0.1 m potassium phosphate, pH 7, containing 10 mm β-ME, and 10 μm Zn2+, which was originally selected because it corresponds to optimal assay conditions for mammalian PBGS. Under these conditions, purified DmPBGS has a specific activity of ∼2.5 μmol h–1 mg–1, which is an order of magnitude lower than human PBGS. Under optimal conditions for DmPBGS (see below), its specific activity is ∼17 μmol h–1 mg–1. Freshly purified DmPBGS contains 1.7 + 0.2 zinc/subunit as determined by atomic absorption spectroscopy. Based on a dry weight analysis, DmPBGS has a 1%A280 = 0.79. Kinetic and Metal Binding Properties of DmPBGS— DmPBGS activity varies as a function of pH as illustrated in Fig. 1 and quantified in Table I. The maximal activity is observed with no added metals and peaks at ∼pH 8. Under these conditions, the pH activity profile shows a good fit to a simple bell curve with pK a = 7.2, pK b = 9.1 and a maximal velocity of 21 μmol h–1 mg–1. The addition of 0.1 m K+ or 1 mm Mg2+, which are required for, or stimulate, the activity of most other PBGS, has little effect on the pK a and pK b values, though Mg2+ appears to afford some inhibition based on V max (see Table I). The addition of 10 μm Zn2+, however, dramatically alters the profile to reveal two pK a values of 6.1 and 10.0, both of which fit best to a two-proton model. The net effect of 10 μm Zn2+ at pH values below the optimal pH of 8 is a dramatic inhibition. For comparison, at its optimal pH of ∼7, human PBGS is not inhibited by Zn2+ at concentrations below ∼30 μm (9Jaffe E.K. Volin M. Bronson-Mullins C.R. Dunbrack Jr., R.L. Kervinen J. Martins J. Quinlan Jr., J.F. Sazinsky M.H. Steinhouse E.M. Yeung A.T. J. Biol. Chem. 2000; 275: 2619-2626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Jaffe E.K. Salowe S.P. Chen N.T. DeHaven P.A. J. Biol. Chem. 1984; 259: 5032-5036Abstract Full Text PDF PubMed Google Scholar). The pH activity profile of human PBGS at 10 μm Zn2+ is included in Fig. 1 and in Table I.Table IQuantitative fit data to the pH rate profiles of DmPBGS and H10F illustrated in Fig. 1PBGS proteinConditionsV max or V max1pKa or pKa 1pKb or pKa 2V max2n1n2DmPBGSaThe fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).No added metals20.51 ± 0.447.19 ± 0.039.05 ± 0.0411DmPBGSaThe fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).0.1 m K+20.27 ± 0.527.09 ± 0.038.92 ± 0.0411DmPBGSaThe fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).1 mm Mg2+16.54 ± 0.577.19 ± 0.049.19 ± 0.0811DmPBGSbThe fitted equation is velocity = (V max1/(1 + 10(2 × (pKa 1 - pH)))) + (V max2/(1 + 10(2 × (pKa 2 - pH)))). Reported pK values are pKa 1 and pKa 2.10 μm Zn2+2.20 ± 0.076.13 ± 0.058.12 ± 0.0110.00 ± 0.1122HumancThe fitted equation is velocity = V max/(1 + 10(2 × (pKa - pH)) + 10(pH - pKb)).10 μm Zn2+46.47 ± 0.795.80 ± 0.048.13 ± 0.0321H10FaThe fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).No metal14.17 ± 0.617.12 ± 0.059.15 ± 0.0511H10FaThe fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).10 μm Zn2+14.00 ± 0.427.06 ± 0.049.24 ± 0.0411a The fitted equation is velocity = V max/(1 + 10(pKa - pH) + 10(pH - pKb)).b The fitted equation is velocity = (V max1/(1 + 10(2 × (pKa 1 - pH)))) + (V max2/(1 + 10(2 × (pKa 2 - pH)))). Reported pK values are pKa 1 and pKa 2.c The fitted equation is velocity = V max/(1 + 10(2 × (pKa - pH)) + 10(pH - pKb)). Open table in a new tab The K m for ALA was determined for DmPBGS at pH 8 with no added metal ions and found to be 108 + 12 μm, which is in the range of all other PBGS at their conditions of optimal pH and metal ions (e.g. 4). The V max was found to be 16.8 + 0.3 μmol h–1 mg–1. The kinetic parameters K m and V max were not quantified under other conditions of pH or buffer metal ion concentrations. Fig. 2 quantifies the effect of adding Zn2+ on the activity of DmPBGS at pH 8 and at 10 mm ALA. The data show inhibition to about 10% of maximal activity, which fit well to Equation 1. Velocity=Vmin+(Vmax-Vmin)/(1+([Zn2+]/IC50)Hillcoefficient)(Eq. 1) The resulting IC50 = 4.24 + 0.49 μm Zn2+, and the Hill coefficient is 1.51 + 0.23. It is interesting to note that the best fit to V min is 1.89 + 0.61 μmol h–1 mg–1, in excellent agreement with the plateau rate observed between pH 6.5 and 7.5 at 10 μm Zn2+ (see Fig. 1 and V max1 in Table I). To quantitatively correlate the inhibitory effect of zinc on enzyme activity with the zinc content of the protein, equilibrium dialysis studies were carried out under assay conditions in the presence and absence of 1 mm ALA in the dialysis buffer. These data are shown in Fig. 3A and quantified in Table II. The data fit well to the following simple binding in Equation 2, ZnTB=Zncat+((Znlnh×[Zn2+])/(KdZnInh+[Zn2+])),(Eq. 2) where ZnTB is total bound zinc, Zncat is the stoichiometry of a required zinc, ZnInh is the stoichiometry of an inhibitory zinc, [Zn2+] is the free Zn2+ concentration, and K dZnInh is the dissociation constant for the inhibitory zinc. Correlation of Fig. 3 with Fig. 2 indicates that under conditions where zinc does not inhibit, there are ∼0.5–0.7 zinc/subunit, which is consistent with half-of-the-sites metal binding. Under conditions where zinc shows significant inhibition of DmPBGS, the protein contains ∼1.5 zinc/subunit, suggesting that the inhibitory zinc has a stoichiometry of 1/subunit. The K dZnInh value for binding the inhibitory zinc in the presence of substrate, ∼7 μm Zn2+, is in good agreement with the kinetically determined IC50 of ∼4 μm Zn2+ (see Fig. 2).Table IIZinc binding and stoichiometry data for DmPBGS and H10FPBGSDialysis conditionsZinc/subunit corresponding to full activityZinc/subunit at maximum bindingKd for looser binding zincIC50 for o-phea[o-phe] required for 50% loss of the catalytic zinc.Hill coefficient for zincμ mmmDmPBGS[Zn2+]0.47 ± 0.031.92 ± 0.3325.4 ± 10.8bApparent K d for inhibitory zinc.1DmPBGS[Zn2+] + ALA0.69 ± 0.021.45 ± 0.066.79 ± 1.54bApparent K d for inhibitory zinc.1DmPBGS[o-phe]0.50 ± 0.040.00 ± 0.040.27 ± 0.061.7 ± 0.64DmPBGS[o-phe] + ALA0.54 ± 0.020.00 ± 0.091.03 ± 0.022.2 ± 1.3H10F[Zn2+]0.52 ± 0.061.18 ± 0.146.47 ± 3.72cApparent K d for nonessential zinc.H10F[Zn2+] + ALA0.54 ± 0.061.01 ± 0.125.72 ± 4.06cApparent K d for nonessential zinc.a [o-phe] required for 50% loss of the catalytic zinc.b Apparent K d for inhibitory zinc.c Apparent K d for nonessential zinc. Open table in a new tab To demonstrate that DmPBGS requires at least some of the tightly bound zinc that is found to co-purify with the protein, DmPBGS activity was assessed as a function of the zinc chelator, o-phe, as illustrated in Fig. 2. Maximal activity is observed at o-phe concentrations below 0.1 mm, and the inhibition profile fits best to a cooperative model where IC50 = 0.84 + 0.03 mm o-phe with a Hill coefficient of 1.9 + 0.1. To determine enzyme-bound Zn2+ as a function of o-phe equilibrium dialysis, experiments were carried out in the presence and absence of 1 mm ALA as illustrated in Fig. 3A and quantified in Table II. At low o-phe the maximal catalytic activity seen in Fig. 2 is again associated in Fig. 3A with Zn2+ bound at a stoichiometry of 0.5/subunit, confirming half-of-the-sites binding of the catalytic Zn2+. In this case, the binding data fit best to a cooperative model for o-phe inhibition (Table II). Based on the IC50 of o-phe (the amount of o-phe required to remove one-half of the catalytic zinc) in the presence and absence of the substrate ALA (1 mm versus 0.3 mm o-phe, respectively), the catalytic zinc appears to be more tightly bound in the presence of ALA than in its absence. This is consistent with a model where ALA provides some of the ligands to this required zinc ion. It is reassuring that the apparent IC50 of o-phe (1 mm) in the presence of ALA is in excellent agreement with the kinetically determined IC50 of o-phe (∼0.8 mm). The data presented in Figs. 2 and 3 show that DmPBGS binds Zn2+ differently from either human or yeast PBGS. Like the ZnB of human PBGS (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), the catalytic Zn2+ of DmPBGS shows half-of-the-sites metal binding. However, Fig. 3A suggests that the inhibitory Zn2+ of DmPBGS does not show the half-of-the-sites binding that has been seen for the non-essential ZnA of human PBGS (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In support of the notion that the inhibitory zinc of DmPBGS is somehow related to ZnA of yeast and mammalian PBGS, the KdZnInh of DmPBGS is of comparable magnitude to the K d for ZnA of mammalian PBGS (∼5 μm), which binds at 0.5 zinc/subunit (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 9Jaffe E.K. Volin M. Bronson-Mullins C.R. Dunbrack Jr., R.L. Kervinen J. Martins J. Quinlan Jr., J.F. Sazinsky M.H. Steinhouse E.M. Yeung A.T. J. Biol. Chem. 2000; 275: 2619-2626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Jaffe E.K. Salowe S.P. Chen N.T. DeHaven P.A. J. Biol. Chem. 1984; 259: 5032-5036Abstract Full Text PDF PubMed Google Scholar). The DmPBGS Mutant H10F—Cys-219 of DmPBGS is analogous to the cysteine that has been shown to bind the nonessential (but also non-inhibitory) ZnA of human (4Jaffe E.K. Martins J. Li J. Kervinen J. Dunbrack Jr., R.L. J. Biol. Chem. 2001; 276: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and yeast (6Erskine P.T. Senior N. Awan S. Lambert R. Lewis G. Tickle I.J. Sarwar M. Spencer P. Thomas P. Warren M.J. Shoolingin-Jordan P.M. Wood S.P. Cooper J.B. Nat. Struct. Biol. 1997; 4: 1025-1031Crossref PubMed Scopus (131) Google Scholar) PBGS. In our model of DmPBGS, shown in Fig. 4A, Cys-219 lies very near to His-10 of a neighboring subunit, such that Cys-219 and His-10 could form part of a previously uncharacterized zinc binding site. Various PBGS crystal structures suggest that the position of Cys-219 is dependent on whether the active site lid is opened or closed. Hence, zinc binding to this site in the closed lid conformation shown in Fig. 4A could inhibit lid opening and thus inhibit catalysis. To test the hypothesis that His-10 is involved in binding the inhibitory zinc, this residue was altered to Phe, which is found in the analogous position of human PBGS. The behavior of DmPBGS mutant H10F during purification was indistinguishable from wild type DmPBGS. The yield was 75 mg from 17 g of cell paste. The specific activity of H10F at pH 8 is seen to be ∼12 μmol h–1 mg–1, only marginally lower than wild type, regardless of whether or not 10 μm Zn2+ was added to the assay mixture. The pH rate profile of H10F does not exhibit the zinc inhibition phenomenon but is otherwise the same as the wild type protein (see Fig. 1). The fitted pH rate profile data is detailed in Table I; however, for clarity of presentation only the combined fit line is illustrated in Fig. 1 (R 2 = 0.96). The behavior of H10F supports the hypothesis that inhibition by zinc is because of zinc binding through His-10 of DmPBGS. DmPBGS mutant H10F provides an independent tool for determining the stoichiometry of the inhibitory zinc of DmPBGS. To do so, the H10F protein was evaluated for its ability to bind zinc in an equilibrium dialysis experiment, the results of which are illustrated in Fig. 3B. In this case the looser binding zinc of H10F retains the K d of ∼5 μm but binds at a reduced stoichiometry of ∼0.5/subunit. Thus, H10F has lost the inhibitory zinc, which appears to have bound at a stoichiometry of 0.5/subunit. The conclusion is that DMPBGS contains the catalytic Zn2+ (akin to ZnB) at 4/octamer, contains the non-essential Zn2+ (akin to ZnA) at 4/octamer, and contains an inhibitory Zn2+ at 4/octamer. Because Cys-219 is implicated in binding both ZnA and the inhibitory zinc, the binding of these two metal ions must be mutually exclusive (see “Discussion”). Sensitivity of DmPBGS to the Species-selective Inhibitors 4,7-DOSA and 4-OSA—The differences in identity and stoichiometry of active site and allosteric metal ions for the different species of PBGS have been found to correlate with a differential susceptibility to various active site-directed inhibitors (7Kervinen J. Jaffe E.K. Stauffer F. Neier R. Wlodawer A. Zdanov A. Biochemistry. 2001; 40: 8227-8236Crossref PubMed Scopus (40) Google Scholar, 8Jaffe E.K. Kervinen J. Martins J. Stauffer F. Neier R. Wlodawer A. Zdanov A. J. Biol. Chem. 2002; 277: 19792-19799Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). This is most notable for the inhibitor 4,7-dioxosebacic acid (4,7-DOSA), which we have previously characterized with PBGS from human, E. coli, P. aeruginosa, Pisum sativum (pea), and B. japonicum, each of which contains a unique constellation of active site and allosteric metal ions. We find that a 100-min preincubation of DmPBGS with 4,7-DOSA over the concentration range 0.1 μm–1 mm reveals an IC50 of 9.7 ± 0.5 μm 4,7-DOSA, with a Hill coefficient of 1.5. This fit is significantly better than the fit to a simple hyperbolic equation, which gives IC50 of 9.1 ± 0.9 μm 4,7-DOSA. The DmPBGS mutant H10F exhibits a comparable IC50 of 9.6 ± 0.5 but fits quite well to a non-cooperative model. Fig. 5A puts 4,7-DOSA inhibition of DmPBGS in the context of prior studies with other species of PBGS (7Kervinen J. Jaffe E.K. Stauffer F. Neier R. Wlodawer A. Zdanov A. Biochemistry. 2001; 40: 8227-8236Crossref PubMed Scopus (40) Google Scholar). The prior work suggested that sensitivity of PBGS to 4,7-DOSA is significantly enhanced by the active site zinc, and results with DmPBGS are consistent with that observation. The species-dependent inhibition pattern observed for 4-OSA was significantly different from that of 4,7-DOSA (8Jaffe E.K. Kervinen J. Martins J. Stauffer F. Neier R. Wlodawer A. Zdanov A.