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• W2169397609 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. 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. porphobilinogen synthase 5-aminolevulinic acid 2-mercaptoethanol 1,3-bis[tris(hydroxymethyl)-methylamino] propane matrix-assisted laser desorption ionization/time of flight Porphobilinogen synthase (PBGS,1 also known as 5-aminolevulinate dehydratase) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen, the monopyrrole precursor of tetrapyrroles (see Fig. 1). Although PBGS proteins from different organisms differ in their use of metal ions, there is remarkable sequence conservation between the PBGS from eubacteria, archaea, and eucaryotes implying a commonality in the overall protein architecture and reaction mechanism (1Jaffe E.K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 115-128Crossref PubMed Scopus (92) Google Scholar). The two PBGS studied herein, those of Escherichia coli and human, both require a catalytic Zn(II), neither require monovalent cations, and the E. coli PBGS activity is stimulated by an allosteric Mg(II) (2Mitchell L.W. Jaffe E.K. Arch. Biochem. Biophys. 1993; 300: 169-177Crossref PubMed Scopus (50) Google Scholar, 3Spencer P. Jordan P.M. Biochem. J. 1993; 290: 279-287Crossref PubMed Scopus (49) Google Scholar, 4Jaffe 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, 5Jaffe E.K. Ali S. Mitchell L.W. Taylor K.M. Volin M. Markham G.D. Biochemistry. 1995; 34: 244-251Crossref PubMed Scopus (77) Google Scholar). The overall protein sequence identity between E. coli and human PBGS is 42%, and the active site residues are significantly more conserved. Considerable effort has been applied to the characterization of PBGS proteins from various organisms, and several crystal structures are now available (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, 7Erskine P.T. Norton E. Cooper J.B. Lambert R. Coker A. Lewis G. Spencer P. Sarwar M. Wood S.P. Warren M.J. Shoolingin-Jordan P.M. Biochemistry. 1999; 38: 4266-4276Crossref PubMed Scopus (83) Google Scholar, 8Frankenberg N. Erskine P.T. Cooper J.B. Shoolingin-Jordan P.M. Jahn D. Heinz D.W. J. Mol. Biol. 1999; 289: 591-602Crossref PubMed Scopus (73) Google Scholar, 9Erskine P.T. Newbold R. Roper J. Coker A. Warren M.J. Shoolingin-Jordan P.M. Wood S.P. Cooper J.B. Protein Sci. 1999; 8: 1250-1256Crossref PubMed Scopus (44) Google Scholar, 10Erskine P.T. Duke E.M. Tickle I.J. Senior N.M. Warren M.J. Cooper J.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 421-430Crossref PubMed Scopus (34) Google Scholar). Yet much of the chemical reaction mechanism remains speculative (7Erskine P.T. Norton E. Cooper J.B. Lambert R. Coker A. Lewis G. Spencer P. Sarwar M. Wood S.P. Warren M.J. Shoolingin-Jordan P.M. Biochemistry. 1999; 38: 4266-4276Crossref PubMed Scopus (83) Google Scholar). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11Nandi D.L. Shemin D. J. Biol. Chem. 1968; 31: 1236-1242Abstract Full Text PDF Google Scholar). Several x-ray crystal structures contain levulinic acid bound in a fashion analogous to the Schiff base (Protein Database codes 1B4K, 1YLV, and 1B4E), and the actual Schiff base involving ALA has been observed by 13C and15N NMR for a chemically modified form of bovine PBGS (12Jaffe E.K. Markham G.D. Biochemistry. 1987; 26: 4258-4264Crossref PubMed Scopus (33) Google Scholar,13Jaffe E.K. Markham G.D. Rajagopalan J.S. Biochemistry. 1990; 29: 8345-8350Crossref PubMed Scopus (38) Google Scholar). This work describes the catalytic and physical properties of PBGS variants with mutations to the Schiff base forming lysine, which for E. coli and human PBGS are Lys-246 and Lys-252, respectively. An appealing aspect of these variants is their expected inability to catalyze porphobilinogen formation and thus there exists the potential of observing new enzyme-bound reaction intermediates using 13C NMR. Although the proteins were found not to be sufficiently inactive for that purpose, the 13C NMR spectra of E. coli Lys-246 mutants with 13C-labeled product bound at the active site are presented. The number of functional active sites reported for the PBGS homo-octamer varies between four and eight, and it remains unclear if there is a unifying thread that will resolve this apparent discrepancy. For instance, data on E. coli PBGS have been interpreted to support both four and eight active sites/octamer (3Spencer P. Jordan P.M. Biochem. J. 1993; 290: 279-287Crossref PubMed Scopus (49) Google Scholar, 14Spencer P. Jordan P.M. Biochem. J. 1994; 300: 373-381Crossref PubMed Scopus (32) Google Scholar, 15Mitchell L.W. Volin M. Jaffe E.K. J. Biol. Chem. 1995; 270: 24054-24059Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 16Senior N.M. Brocklehurst K. Cooper J.B. Wood S.P. Erskine P. Shoolingin-Jordan P.M. Thomas P.G. Warren M.J. Biochem. J. 1996; 320: 401-412Crossref PubMed Scopus (55) Google Scholar), yet the data on mammalian PBGS uniformly supports four functional active sites (17Bevan D.R. Bodlaender P. Shemin D. J. Biol. Chem. 1980; 255: 2030-2035Abstract Full Text PDF PubMed Google Scholar, 18Jaffe E.K. Salowe S.P. Chen N.T. DeHaven P.A. J. Biol. Chem. 1984; 259: 5032-5036Abstract Full Text PDF PubMed Google Scholar, 19Jordan P.M. Gibbs P.N. Biochem. J. 1985; 227: 1015-1020Crossref PubMed Scopus (45) Google Scholar, 20Jaffe E.K. Abrams W.R. Kaempfen H.X. Harris Jr., K.A. Biochemistry. 1992; 31: 2113-2123Crossref PubMed Scopus (46) Google Scholar). The present active site lysine mutants further probed this apparent anomaly. ALA-HCl, KPi, Bis-tris propane, and p-(dimethylamino)-benzaldehyde were from Sigma. 2-Mercaptoethanol (βME) was from Fluka (Ronkonkoma, NY) and distilled under vacuum prior to use. HgCl2, ZnCl2, MgCl2 (ultrapure), and high purity KOH were from Aldrich. [4-13C]ALA was custom-synthesized by C/D/N Isotopes (Pointe-Claire, Quebec, Canada). [4-14C]ALA (50 μCi/μmol) and [3H]ALA (1.6 Ci/mmol) were from Amersham Pharmacia Biotech. Centrifree and Centriprep ultrafiltration devices were from Amicon Corp. (Danvers, MA). Slide-A-Lyser devices were from Pierce. House distilled water was further purified by passage through a Milli-Q water purification system (Millipore, Bedford, MA). DNA plasmid purification kits were from Qiagen (Valencia, CA). Oligonucleotides were synthesized in-house by the Fannie Ripple Biotechnology Center. All other chemicals were reagent grade. PBGS activity assays were as described previously for E. coli (2Mitchell L.W. Jaffe E.K. Arch. Biochem. Biophys. 1993; 300: 169-177Crossref PubMed Scopus (50) Google Scholar) and human PBGS (4Jaffe 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 standard assay buffer contained 0.1m KPi, pH 7.0, 10 mm βME, 10 μm Zn(II), and ∼5–15 μg ml−1 PBGS. E. coli PBGS assays also contained 1 mm Mg(II). The standard assay is started by the addition of ALA-HCl to a final concentration of 10 mm, which lowers the pH to 6.8. After 5 min the reaction is quenched, and porphobilinogen is determined using Ehrlich's reagent. Up to 1 mg ml−1 of purified PBGS and a 15-min incubation were used for mutant proteins. The plasmid pCR261, containing the E. coli hemBgene for PBGS, was a kind gift of Dr. C. Roessner of Texas A & M, College Station, TX (21Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar). A 1400-base pair fragment containing the 1100-base pair hemB gene was excised from the EcoRI and BamHI site of pCR261 and inserted into the multiple cloning site of pUC119 to form pLM1228. The Muta-Gene kit from Bio-Rad was used for site-directed mutagenesis on hemBas described previously (15Mitchell L.W. Volin M. Jaffe E.K. J. Biol. Chem. 1995; 270: 24054-24059Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) using the primers listed in Table I. The plasmids, pK246N, pK246G, pK246W, pK246H, and pK246M, were transformed into the E. coli strain HB101; the plasmid sequences were confirmed throughout the entire hemB gene by the FCCC DNA Sequencing Facility such that two independent complementary data sets were obtained for all coding sequences. All plasmid preparations used for sequencing were derived directly from bacterial growths used for protein preparations. All plasmids contained the expected mutation and no second site mutations. The sequence agreed with the hemBgene sequence derived from the 6′-8′ region of the E. coligenome (GenBankTM accession number U73857). There are significant differences between these and earlier published hemB gene sequences (22Echelard Y. Dymetryszyn J. Drolet M. Sasarman A. Mol. Gen. Genet. 1988; 214: 503-518Crossref PubMed Scopus (33) Google Scholar, 23Li J.M. Russell C.S. Cosloy S.D. Gene (Amst.). 1989; 75: 177-184Crossref PubMed Scopus (32) Google Scholar). Mutations to human PBGS were obtained using the QwikChange technology of Stratagene on the plasmid pMVhum as described previously (4Jaffe 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 sense strand of the mutagenic primer for K252G was CGCAGACATGCTGATGGTTGGACCTGGAATGCC.Table ICharacteristics of E. coli PBGS and Lys-246 variantsPBGSMutagenesis informationSpecific activityEquilibrium binding of Zn(II) and Mg(II)Mass spectral data for the Asp-241–Leu-252 proteolytic fragment13C NMR chemical shifts for enzyme-bound [3,5-13C]porphobilinogenMutagenic primers directed toward non-coding strandNew restriction siteAt 10 μm ZnAt 30 μm ZnPredicted massObserved massC-3 ppmC-5 ppmMg/subunitZn/subunitMg/subunitZn/subunitμmol h −1 mg −1Wild-type500.970.801.00.931338.61338.4121.4127.3K246NAAA → AACHpaI0.0800.940.791.00.941324.51324.7122.5128.5K246GAAACCT → GGGCCGNgoMI0.0101.010.801.00.921267.51266.7123.9128.5K246MAAACCT → ATGCCGNgoMI0.0700.970.811.00.941341.61341.4123.4127.3K246HGTTAAA → GTGCACApaLI0.0230.950.781.10.941347.61346.6123.3128.7K246WAAACCT → TGGCCGNgoMI0.0950.960.801.00.941396.61396.1NANAFree porphobilinogen123.1121.0 Open table in a new tab The constructs HB101(pLM1228), HB101(pK246N), HB101(pK246G), HB101(pK246W), HB101(pK246H), and HB101(pK246M) gave excellent constitutive expression of mutant or wild-type E. coli PBGS at 15–30% the total soluble cellular protein. In an unsuccessful attempt to obtain inactive protein not contaminated with chromosomally encoded wild-type E. coli PBGS, the plasmids pK246N, pK246G, pK246H, pK246M, and pK246W were each transformed into the hemB − strains RP523 or HU1000 in the presence of hemin. HU1000 (hemB, thr1, leuB6, thi1, lacY1, tonA21, suppE44, λ−, F−) and its parent strain RP523, the kind gifts of Drs. C. Russell and S. Cosloy of City College of New York, do not express a functional PBGS, require hemin, and are hemin-permeable (which most E. coli are not) (24Umanoff H. Russell C.S. Cosloy S.D. J. Bacteriol. 1988; 170: 4969-4971Crossref PubMed Google Scholar). Complementation was determined as described previously (15Mitchell L.W. Volin M. Jaffe E.K. J. Biol. Chem. 1995; 270: 24054-24059Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The transformants proved unstable, and it was not possible to obtain expression of inactive recombinant PBGS from these constructs, even when the bacteria were grown fermentatively to minimize the metabolic requirement for tetrapyrroles. Human PBGS was expressed as described previously (4Jaffe 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) from an artificial gene contained in BLRDE3(pMVhum) or BLRDE3(pK252G). This human PBGS corresponds to the most abundant of two common alleles and contains a lysine at position 59 (K59). Purification of wild-type E. coli PBGS was as described previously (2Mitchell L.W. Jaffe E.K. Arch. Biochem. Biophys. 1993; 300: 169-177Crossref PubMed Scopus (50) Google Scholar, 5Jaffe E.K. Ali S. Mitchell L.W. Taylor K.M. Volin M. Markham G.D. Biochemistry. 1995; 34: 244-251Crossref PubMed Scopus (77) Google Scholar). All purification buffers contained 10 μm Zn(II) and 1 mm Mg(II). Yields were ∼15 mg of protein/g of cells. All five mutant proteins were purified from transformants of strain HB101. In these cases, the crude extract was subject to a 45% ammonium sulfate precipitation, and the pellet was redissolved and run through a Sephacryl S-300 column; the major protein peak at 41–46% column volume was then purified on a DE-Biogel A column, and resultant protein was concentrated and stored at −70 °C. Some NMR studies of protein samples to which substrate was added revealed the time-dependent loss of porphobilinogen and formation of an alternative pyrrole thus indicating a contaminant protein that metabolized porphobilinogen. This contaminant could be removed by a second passage through the S-300 column. Based on an assay of background levels of PBGS expressed by HB101, mutant protein preparations from constructs in HB101 are contaminated with endogenous wild-type PBGS encoded by the hemB gene of E. coli strain HB101 at a level of 0.02–0.1%. Human PBGS corresponding to the K59 allele and the K252G variant were purified as described previously where the DEAE column provided a separation of the Mg(II)-responsive and chromosomally encoded E. coli PBGS from Mg(II)-insensitive human PBGS (4Jaffe 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). E. coli PBGS (wild-type and Lys-246 variants) at 2 mg ml−1 were incubated at 37 °C for 10 min in 0.1 m KPi, pH 7, 10 mm βME, 10 μm Zn(II), and 1 mmMg(II) (Buffer I). The proteins were then placed on ice for ∼5 min followed by the addition of 1.9 mg of NaBH4 and 1 min later by the addition of [4-14C]ALA (9 × 104cpm/μmol) to a final concentration of 10 mm ALA-HCl. Controls were included in which water replaced NaBH4 and 0.1 n HCl replaced the stock 0.1 m ALA-HCl. After 10 min, the reactions were quenched by the addition of 3 volumes of ice-cold, saturated (NH4)2SO4and allowed to sit for 30 min. The precipitated protein was centrifuged, and the protein pellets were redissolved in ∼1 ml of Buffer I. The protein samples were then dialyzed overnight versus 4 liters of Buffer I prior to radioactivity, protein concentration, and catalytic activity determinations. Schiff base trapping studies on human PBGS used unlabeled ALA and followed the same protocol with the exception that Mg(II) was omitted from Buffer I. E. coli PBGS and all the Lys-246 variants (∼200 μg) were digested overnight using 2 μg of AspN protease after cysteine residues were modified by iodoacetamide. The resultant peptides were mapped by reverse phase high pressure liquid chromatography as described previously (20Jaffe E.K. Abrams W.R. Kaempfen H.X. Harris Jr., K.A. Biochemistry. 1992; 31: 2113-2123Crossref PubMed Scopus (46) Google Scholar). Peaks were collected manually. “Matching” peaks were identified by inspection of the peptide map. All matching peaks as well as major “additional peaks” were characterized by MALDI-TOF mass spectral analysis using a Perseptive Voyager DE instrument. The program PAWS (R. C. Beavis, New York University) was used to help identify the various peptides. For identification of the Schiff base containing peptide, high specific radioactivity [3H]ALA and NaBH4 were used as above to label the reactive lysine; then the Schiff base-modified peptide was identified following AspN protease digestion. The amino acid sequence of the radiolabeled peptides was obtained using Edman degradation techniques by W. R. Abrams in the Protein Analytical Laboratory at the University of Pennsylvania School of Dental Medicine. Atomic absorption analysis of E. coli PBGS for Zn(II) and Mg(II) were done on a PerkinElmer Life Sciences AAnalyst 100 spectrometer using an air/acetylene flame. Slide-A-Lysers containing each PBGS variant, ∼0.2 ml at 25–35 mg ml−1, were dialyzed together against 2 liters of 0.1 m Bis-tris propane, pH 8, containing 10 mm βME, 1 mmMg(II), and either 10 μm or 30 μm Zn(II). The dialysate was analyzed for Zn(II) directly without dilution. The dialysate was diluted 50-fold prior to analysis for Mg(II) to be within the linear range of the instrument. The protein samples were each diluted ∼50-fold prior to analysis for Zn(II), Mg(II), and protein. E. coli PBGS samples, at concentrations of both 7 mg ml−1 (0.2 mm subunits) and 25–35 mg ml−1(∼1 mm subunits) in ∼250-μl volumes, were placed in Slide-A-Lysers (0.5 ml maximum volume) and equilibrated by dialysis at room temperature against 40 ml of 0.1 m Bis-tris propane, pH 8, 10 mm βME, 10 μm Zn(II), 1 mm Mg(II), and various concentrations of porphobilinogen ranging from 5 μm to 1.4 mm. The concentration of porphobilinogen inside and outside the Slide-A-Lyser was determined using Ehrlich's reagent. Protein concentrations were determined by quantitative total amino acid analyses using vapor phase hydrolysis in 6 n HCl, 0.1% phenol, at 110 °C for 22 h followed by phenylisothiocyanate pre-column derivatization and high pressure liquid chromatography analysis. Spectral parameters were as described previously (12Jaffe E.K. Markham G.D. Biochemistry. 1987; 26: 4258-4264Crossref PubMed Scopus (33) Google Scholar). The E. coli protein samples uniformly contained 100–150 mg of PBGS in 1.8 ml (∼1 mm active sites) of buffer containing 20% D2O. The buffers were 0.1m KPi or Bis-tris propane-HCl, 10 mm βME, 1 mm Mg(II), 10 μmZn(II) at pH 6, 7, or 8. In all cases, natural abundance13C NMR spectra of the proteins were obtained (12,000–36,000 scans) prior to the addition of [4-13C]ALA. Following addition of [4-13C]ALA, all samples catalyzed the stoichiometric conversion to [3,5-13C]porphobilinogen. Dialysis at pH 6 was used to remove enzyme-bound product after the enzyme-product spectra were obtained. The chemical shifts of [3,5-13C]porphobilinogen (both free and bound) are insensitive to these changes in buffer or pH. However, the Kd for the enzyme-product complex is highly pH-dependent (15Mitchell L.W. Volin M. Jaffe E.K. J. Biol. Chem. 1995; 270: 24054-24059Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Spectra obtained after dialysis at pH 6 confirmed the dissociation of enzyme-bound product. The E. coli PBGS proteins K246M, K246W, K246H, K246G, and K246N as purified from the E. coli host strain HB101 have specific activities ranging from 0.01 to 0.1 μmol h−1mg−1, compared with the wild-type enzyme value of ∼50 μmol h−1mg−1 (Table I). Thus, these mutants are remarkably impaired in their catalytic potency. Some proportion of the activities observed in these mutants was determined to be due to variable levels of the chromosomally encoded wild-type PBGS because this activity can be reduced significantly by treatment with ALA and NaBH4 (see below). The mutant protein K246G was designed to contain a “hole” at the active site into which the lysine side chain surrogate ethylamine might bind and restore activity. Addition of 50 mm ethylamine had no effect on the specific activity of the K246G preparation or wild-type PBGS and did not alter the chemical shifts of [3,5-13C]porphobilinogen bound to K246G (see below). The purification properties of all the mutant proteins did not differ appreciably from wild-type E. coli PBGS protein. All were stable, highly soluble, octameric proteins. Attempts to obtain expression of inactive Lys-246 mutants in the hemB − hosts, RP523 and HU1000, either failed or resulted in recombination events that regenerated wild-type protein. Human PBGS and the K252G mutant were purified from a BLR(DE3) host E. coli strain (4Jaffe 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). Fig. 2shows that the chromosomally encoded E. coli PBGS activity separates from the relatively inactive human PBGS K252G variant during the DEAE chromatography step used during purification. Whether the residual activity is due to E. coli PBGS can be shown by its ability to be activated by Mg(II). The low activity of the final purified K252G from the S-300 column showed no activation upon addition of Mg(II). The purified human PBGS K252G variant showed a Km of 5.6 mm and a Vmax of 0.026 μmol h−1 mg−1 relative to the wild-type values for K59 of 0.1 mm and 44 μmol h−1 mg−1 for Km and Vmax, respectively, giving a 105-fold reduction in V/K. In this case, the low level of activity was not sensitive to inactivation through treatment with ALA and NaBH4 (see below). Thus, we can exclude the possibility that the activity is due to either 1) translational error or 2) incorporation of E. coli PBGS monomers into the homo-octameric human PBGS. Human PBGS K252G activity is also sensitive to inhibition by Pb(II) which indicates that the alternative Lys-252 Schiff base independent mechanism depends on the catalytic Zn(II) (25Jaffe E.K. Martins J. Kervinen J. Li J. Dunbrack Jr., R.L. J. Biol. Chem. 2000; 275: 1531-1537Abstract Full Text Full Text PDF Scopus (35) Google Scholar). The binding of Zn(II) and Mg(II) to E. coliPBGS is cooperative (5Jaffe E.K. Ali S. Mitchell L.W. Taylor K.M. Volin M. Markham G.D. Biochemistry. 1995; 34: 244-251Crossref PubMed Scopus (77) Google Scholar, 13Jaffe E.K. Markham G.D. Rajagopalan J.S. Biochemistry. 1990; 29: 8345-8350Crossref PubMed Scopus (38) Google Scholar). To address the role of Lys-246 in this cation binding process, we analyzed the ability of the Lys-246 variants to bind Zn(II) and Mg(II). For all five mutants, atomic absorption analysis showed binding of Zn(II) and Mg(II) at the same stoichiometry and affinity as wild-type PBGS (see Table I). Thus, Lys-246 is not functional in the cooperativity of Zn(II) and Mg(II) binding. Because the mutants are not defective in metal binding, this supports an alternative E. coli PBGS reaction mechanism involving the catalytic Zn(II) but lacking the Schiff base to Lys-246. All the E. coliproteins in this study were subjected to Schiff base trapping using radiolabeled ALA and NaBH4. Controls were carried out in the absence of ALA or NaBH4 and both reagents. The results are presented in Table II. For wild-type E. coli PBGS, the control samples, which included all the physical manipulations to the protein, retained complete catalytic activity. In contrast, when treated with both [4-14C]ALA and NaBH4, three samples of wild-type PBGS each showed ∼80% inactivation and 14C labeling at 0.42–0.49 ALA/subunit. This stoichiometry is consistent with four functional active sites per octamer. The mutants K246N, K246G, K246H, K246M, and K246W were not significantly radiolabeled under these conditions; however, in mutants for which activity was above detection levels, the trace activities were significantly reduced, indicating that the activity of these preparations derives from contamination by chromosomally encoded wild-type E. coli PBGS.Table IIALA-dependent NaBH 4 inactivation and [4- 14 C]ALA labeling of PBGSEnzymePercent remaining activity without NaBH4Percent remaining activity with NaBH42-aControls with NaBH4without substrate all gave greater than 95% recovery of activity.14C per octamer with NaBH42-bControls using [4-14C]ALA without NaBH4 all labeled at less than 0.05 per octamer.Wild type E. coli9414–193.36, 3.44, 3.922-cThree separate determinations.K246N92190.08K246G100≤472-dAssay results at detection limit.0.08K246WND2-eND, not determined.ND0.24K246MNDND0.16K246HNDND0.24Wild-type human (K59)9935NDK252G8585ND2-a Controls with NaBH4without substrate all gave greater than 95% recovery of activity.2-b Controls using [4-14C]ALA without NaBH4 all labeled at less than 0.05 per octamer.2-c Three separate determinations.2-d Assay results at detection limit.2-e ND, not determined. Open table in a new tab Human PBGS K59 and K252G were subjected to Schiff base trapping studies using unlabeled ALA. In this case, 83% inactivation was observed for K59, and no inactivation was observed for the K252G variant, which indicates that the activity observed for the latter does not depend upon Schiff base formation between ALA and the protein. Prior studies on mammalian PBGS uniformly show >85% inactivation at stoichiometries approaching 0.5 NaBH4 trapped [14C]ALA per subunit (11Nandi D.L. Shemin D. J. Biol. Chem. 1968; 31: 1236-1242Abstract Full Text PDF Google Scholar, 26Jaffe E.K. Hanes D. J. Biol. Chem. 1986; 261: 9348-9353Abstract Full Text PDF PubMed Google Scholar). AspN protease digestion followed by peptide mapping was carried out for all the E. coli proteins under study. In cases without radioactivity, the identity and sequence of the peptides were deduced from the masses determined by MALDI-TOF analysis. Some peptides were found to result from N-terminal cleavage at glutamic acid. The Lys-246 containing peptide (retention time, ∼52 min, see “Experimental Procedures”) was confirmed by sequencing. In the case of K246M and K246W, the retention time of this peak moved in a predictable fashion as calculated by the program PEPTIDESORT of the GCG package (+10.3 min and + ∼20 min, respectively). For K246G, K246H, and K246N, the match between the predicted and observed variation in retention time was less precise. The observed changes were +9.4 min, −0.4 min, and −4.9 min (predicted times were +2.7, +4.0, and 1.6 min, respectively). As illustrated in Table I, the peptide masses confirmed the identity of the mutation. For wild-type E. coli PBGS treated with tritiated ALA and NaBH4, the radioactivity was found in a cluster of peptide peaks in" @default.
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• W2169397609 title "Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase" @default.
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