©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Bradyrhizobium japonicum Porphobilinogen Synthase Uses Two Mg(II) and Monovalent Cations (*)

(Received for publication, November 8, 1995; and in revised form, January 30, 1996)

Robert M. Petrovich Samuel Litwin Eileen K. Jaffe (§)

From the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bradyrhizobium japonicum porphobilinogen synthase (B. japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M. R. (1995) J. Biol. Chem. 270, 19823-19827). B. japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic Mg(A) present at a stoichiometry of 4/octamer, an allosteric Mg(C) present at a stoichiometry of 8/octamer, and a monovalent metal ion, K. However, the divalent Mg(B) or Zn(B) present in some other PBGS is not present in B. japonicum PBGS. Under optimal conditions, the K for Mg(A) is <0.2 µM, and the K for Mg(C) is about 40 µM. The response of B. japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH. B. japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs.

B. japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic Zn(A). However, consistent with prior predictions, B. japonicum PBGS can bind Zn(II) (presumably as Zn(A)) at a stoichiometry of 4/octamer with a K of 200 µM; but this high concentration is outside a physiologically significant range.


INTRODUCTION

Porphobilinogen synthase (PBGS, (^1)also known as 5-aminolevulinate dehydratase, E.C. 4.2.1.24) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA) to form porphobilinogen as illustrated in Fig. 1(1) . This reaction is common to tetrapyrrole biosynthesis in all phyla (e.g. porphyrin, chlorophyll, corrin, and F430) and is essential for all cellular life. The PBGS octamer contains four active sites, each of which binds two molecules of ALA that have different chemical fates. Although many details of the reaction mechanism are not well established, it is known that there is a Schiff base formed between a universally conserved lysine and one of the two ALA molecules at the active site of the enzyme from all sources examined(2, 3, 4, 5, 6, 35) . The universal Schiff base and high overall sequence conservation suggest that all PBGSs use a common catalytic mechanism(7) . Nevertheless, there are well documented differences in metal ion requirements between evolutionarily divergent PBGS (for example, see (8) ).


Figure 1: The PBGS-catalyzed asymmetric condensation of two molecules of ALA. A-side ALA becomes the acetyl-containing half of PBG and retains the primary amino group. P-side ALA (trapezoid) becomes the propionyl containing half of PBG; its amino group is incorporated into the pyrrole ring.



Until recently, PBGSs from various sources were generally described as requiring either Mg(II) or Zn(II). However, characterization of Escherichia coli PBGS demonstrates that this enzyme can bind both Zn(II) and Mg(II) at different sites and with different functions(9, 10) . Based on studies of E. coli PBGS and mammalian PBGS, this laboratory proposed a model in which any given PBGS had up to three different types of divalent metal ion binding sites called A, B, and C. The function of the divalent ion in site A is to facilitate A-side ALA binding and reactivity (see (7) ). The B-site metal ion has not been found to be ``essential'' although it has been proposed to aid in the removal of a proton lost during porphobilinogen formation(11) . Four A-site metal ions and four B-site metal ions bind to each octamer(12, 13, 14) , equivalent to the stoichiometry of active sites. The C site metal is an allosteric activator that increases V(max), decreases the K for ALA, and decreases the Kfor metal ions in sites A and B; its stoichiometry is 8/octamer(9) . Table 1is an update of our model for divalent metal ion interactions with PBGS. It defines mammalian-like PBGS as type I, containing four Zn(A) and four Zn(B). E. coli-like PBGS is type II; it contains four Zn(A), four Zn(B), and eight Mg(C). (^2)Plant-like PBGS is proposed as type III based on Mg(II) stoichiometry data and an apparent insensitivity of the activity to Zn(II) (16) ; (^3)it hypothetically contains four Mg(A), four Mg(B), and eight Mg(C), although this remains unproven.



The plant endosymbiot Bradyrhizobium japonicum produces a PBGS (B. japonicum PBGS) protein with a hybrid sequence in a putative metal binding domain of PBGS, shown in Fig. 2(17, 18) . To help explain the stoichiometry of sites A and B, 4 sites/homooctamer, this domain of each subunit has been proposed to provide ligands to either the A-site or B-site metal ions(13, 14) . The C site metal ions bind elsewhere in the sequence(20) .^3 The sequence of B. japonicum PBGS in this region is different from those of types I, II, and III PBGS and may define a type IV PBGS. To explore the existence of a type IV PBGS, we purified and characterized B. japonicum PBGS cloned and expressed in E. coli(21) . We find that B. japonicum PBGS represents a type IV PBGS whose divalent metal ions are listed in Table 1and reveals an additional monovalent cation interaction.


Figure 2: A putative metal-binding region of PBGS from 24 species (see (7) ). The cysteine- and histidine-rich sequence of type I (e.g. Homo sapiens) and type II (e.g. E. coli) PBGS is purported to bind Zn(II)(17) . It is replaced by an aspartate-rich region in type III (Pisum sativum) PBGS, purported to bind Mg(II)(16) . B. japonicum (type IV) has a hybrid sequence (18) and is the subject of this study. The sequences are followed by their GenBank accession numbers.




EXPERIMENTAL PROCEDURES

Materials

The chemicals KCl, KP(i), phenylmethylsulfonyl fluoride, ampicillin, chloramphenicol, TES, trichloroacetic acid, p-dimethylaminobenzaldehyde, dithiothreitol, ALA, isopropyl-beta-D-thiogalactopyranoside, bis-tris propane and EDTA (free acid), were all purchased from Sigma, and were ACS reagent grade or better. KOH and ZnCl(2) were purchased from Aldrich. Ultrapure MgCl(2) was purchased from Johnson Matthey. Glacial acetic acid and 70% perchloric acid were purchased from Fischer and were ACS reagent grade. HgCl(2) was purchased from J. T. Baker. 2-Mercaptoethanol was purchased from Fluka and vacuum-distilled prior to use. Ultrapure ammonium sulfate was purchased from ICN Biomedical. Phenyl-Sepharose was purchased from Pharmacia Biotech Inc. DEAE-Biogel P6 was purchased from Bio-Rad. Microcon-10, Centricon-10, and Centriprep-10 concentration devices were purchased from Amicon. Slide-A-Lyzer cassettes were purchased from Pierce.

PBGS Activity Assays

The PBGS activity assay (unless indicated otherwise) included a 10-min preincubation at 37 °C of 34 µg of protein (0.88 µM subunit) in 900 µl of 0.1 M KP(i), pH 7.0, 10 mM MgCl(2), and 10 mM 2-mercaptoethanol. (^4)The addition of 100 µl of 0.1 M ALA-HCl started the reaction and adjusted the final pH to 6.9. After 5 min, the reaction was stopped by the addition of 500 µl of 20% (w/v) trichloroacetic acid containing 0.1 M HgCl(2) (STOP reagent). Quenched reactions were then mixed with an equal volume of Ehrlich's reagent, and the pink color was read at 555 nm. In many cases it was necessary to dilute the quenched reaction mixture up to 5-fold with a 2:1 mixture of buffer and STOP reagent prior to the addition of Ehrlich's reagent to ensure an A leq1. Above an absorbance of 1, the Ehrlich's method is not accurate. For very low activity assays, the reaction was allowed to proceed for up to 5 h. The enzyme is stored in 0.1 M KP(i) buffer at pH 7.0 containing 1 mM MgCl(2) and 10 mM 2-mercaptoethanol. Some early preparations also contained 10 µM Zn(II). For assays performed at very low Mg(II) or very low K, a 3-ml sample of 10.4 mg/ml B. japonicum PBGS was dialyzed for 16 h at 4 °C against 1 liter of 0.1 M bis-tris propane, pH 7.0, containing 0.1 mM Mg(II) with the Slide-A-Lyzer dialysis system. Specific activity is defined as µmol of porphobilinogen formed per h per mg of protein.

Atomic Absorption Analyses

Atomic absorption analysis was performed on a Perkin-Elmer 2380 flame spectrometer at the University of Pennsylvania School of Dental Medicine. For Mg(II) binding studies at mM levels of Mg(II), the atomic absorption samples were prepared by mixing 6.8 mg of enzyme with 1, 3, or 10 mM MgCl(2) and 10 mM 2-mercaptoethanol in the presence of 10 µM or 500 µM ZnCl(2), in a total volume of 230 µl of 0.1 M KP(i). Immediately after mixing, the free and bound metals were separated by ultrafiltration to 100 µl with Microcon-10 concentrators. Both the effluent and the enzyme solutions were then diluted 100-fold in 0.1 M buffer at pH 7.0 for atomic absorption analysis. Standards were prepared in the same buffer. Under conditions of µM Mg(II), atomic absorption samples were prepared by mixing 1.2 mg of enzyme containing 10 mM 2-mercaptoethanol in 3 ml of 0.1 M buffer with 0-50 µM MgCl(2), in the presence of 0 or 10 mM ALA. Free metal was separated from bound metal by ultrafiltration to 1.5 ml using Centricon-10 concentrators; both concentrate and effluent were read directly.

Protein Expression and Purification

The gene for B. japonicum PBGS was a kind gift from Dr. Mark O'Brian of SUNY Buffalo, who provided the clone in the E. coli expression system BL21(DE3)pLysS:pETBJHEMB, which places the hemB gene (encoding B. japonicum PBGS) under the control of the T7 promoter(21) . BL21(DE3)pLysS:pETBJHEMB cells were grown in LB media containing ampicillin and chloramphenicol at 37° C to an A of 1 and then induced by the addition of 0.4 mM isopropyl-beta-D-thiogalactopyranoside. The bacterial cultures were allowed to incubate overnight (16 h) and harvested the next day.

The BL21(DE3)pLysS:pETBJHEMB construct typically yields a crude extract that has greater than 20% of the soluble protein as B. japonicum PBGS. Cells were disrupted by suspending 20 g of cell paste in 40 ml of lysozyme buffer (50 mM KP(i), pH 7.0, 5 mM EDTA, 170 mM KCl, 10 mM 2-mercaptoethanol, and 0.1 mM phenylmethylfulfonyl fluoride) for 1 h at 4 °C during which time the plasmid-encoded lysozyme acts. This was followed by the addition of 40 ml of lysing buffer (0.1 M KP(i), pH 7.0, 12 mM MgCl(2), 40 µM ZnCl(2), 10 mM dithiothreitol, and 0.1 mM phenylmethylfulfonyl fluoride). The suspended cells were further disrupted by three rounds of rapid freeze/thaw followed by sonication. After centrifugation the supernatant (specific activity = 11.8) was subjected to a 30-45% ammonium sulfate fractionation. The 45% pellet was resuspended in 20 ml of 30 mM KP(i) buffer at pH 7.0 containing 113 g/liter ammonium sulfate (20% saturation, loading buffer) and 1 mM MgCl(2), and then loaded onto a 100-ml phenyl-Sepharose column. The column was washed with a linear gradient of loading buffer to 2 mM KP(i) buffer at pH 7.5 containing 1 mM MgCl(2) and 10 mM 2-mercaptoethanol (elution buffer). The enzyme was then eluted from the phenyl-Sepharose column by washing with the elution buffer. Chromosomal encoded E. coli PBGS elutes before B. japonicum PBGS. The protein peak that comes off in the phenyl-Sepharose column in the elution buffer is loaded directly onto a 100-ml DEAE Bio-gel P-6 anion exchange column. The enzyme was eluted from the DEAE column as a single peak using a linear gradient from 0 to 0.5 M KCl in 0.1 M KP(i) buffer at pH 7.5 containing 1 mM MgCl(2) and 10 mM 2-mercaptoethanol. The protein elutes at 250 mM KCl and has a specific activity of 39.1. The overall yield for the purification is 63%. N-terminal amino acid sequence and total amino acid analysis was carried out by the Protein Analytical Laboratory at the University of Pennsylvania School of Dental Medicine.

Data Analysis

Most data were fitted to standard or cited equations using the programs Enzfitter (Elsevier, Biosoft) and KaleidaGraph (Synergy Software). The protein dissociation data were fitted to equations derived in house as follows. The dynamic equilibrium between subunits and multimers is defined by the following reaction,

where S is a subunit and S(n) is a multimer of n subunits. The total concentration of subunits, C, is defined as the sum of all subunits in all species as follows, where N(n) is the concentration of n multimers present.

Expressing C as a function of N(8) and the K(d) values yields the following.

Newton's method was used to calculate N(8) as a function of C(22) . The specific activity (SA) was calculated as follows, where alpha is a theoretical V(max).

These equations allowed us to determine specific activities for each concentration C and any set of constants K, K, K, and alpha. The space of these four constants was searched to fit the calculated specific activities to the observed values by the least squares method.


RESULTS

Working with B. japonicum PBGS we found several unexpected properties of the enzyme that had to be investigated before we could pursue studies of divalent cation effects on B. japonicum PBGS. For instance, during protein purification enzyme assays were plagued by activity fluctuations; these turned out to arise in part from a protein concentration dependence of B. japonicum PBGS activity. Studies designed to define the optimal pH for activity assays revealed an apparent buffer effect, which was determined to be a shift in an essential pK(a) caused by monovalent cations. These properties are described first because they dictate the conditions used for the divalent cation studies.

Protein Purification and Analysis

B. japonicum PBGS exhibits purification characteristics like those of other PBGS. Chromatography on a Sephacryl-300 column yields a profile identical to octameric type I and type II PBGS. N-terminal analysis yielded the sequence AIKYGRPIELREVSR, consistent with that predicted by the cDNA sequence. Total amino acid analysis agreed with that predicted from the cDNA sequence to within an average of 7 ± 6%. The molecular mass determined by mass spectral analysis on a Vestec Malditof (38,710 daltons) is in good agreement with the predicted mass of 38,510 daltons and indicated that the protein was at least 95% pure. When protein is stored at 30 mg/ml in KP(i) buffer containing 1 mM Mg(II), it is stable at 4 °C for at least 1 month. B. japonicum PBGS stored in the absence of Mg(II) and K is prone to precipitate over several days at 4 °C.

Activity as a Function of Protein Concentration

The specific activity of B. japonicum PBGS is protein concentration-dependent; this property has not been found for type I and type II PBGS and most other ``well behaved'' enzymes. As illustrated in Fig. 3A, the activity of B. japonicum PBGS is not linear with protein concentration below 0.9 µM expressed in units of subunit, suggesting a dynamic equilibrium between active multimers and smaller inactive species. The data are consistent with a simple model where monomers are inactive and dimers, tetramers, and/or octamers are active and contain activity proportional to the number of active sites. The solid line in Fig. 3A represents the best fit to this model where the monomer to dimer K is 0.054 ± 0.004 µM; the dimer to tetramer K is 0.55 ± 0.34 µM; and the tetramer to octamer K is 0.013 ± 0.014 µM. This solution predicts that the predominant species are monomer, dimer, and octamer and lends itself to future experimental verification.


Figure 3: Protein concentration- and monovalent cation-dependent activity of B. japonicumPBGS. A, B. japonicum PBGS specific activity as a function of subunit concentration under standard assay conditions (bullet). The solid line represents a model where monomer is inactive and dimer, tetramer, and octamer have activity proportional to the number of active sites. The predicted mol % of monomer, dimer, tetramer, and octamer present at any given concentration of subunits are represented by the (- - -),(- - -), (- bullet -), and (- bullet bullet bullet -) lines, respectively. B, the pH dependence of B. japonicum PBGS in a variety of buffers, which vary in monovalent cation concentration: 0.1 M bis-tris propane-HCl (circle, bullet); 0.1 M TES-KOH (box, ); 0.1 M KP(i) (up triangle, ). Open symbols reflect assays done at 1 mM Mg(II); closed symbols reflect assays done at 10 mM Mg(II). In assays done in bis-tris propane, low levels of K, 2 mM derive from the stock protein solution. C, the pH dependence of B. japonicum PBGS in bis-tris propane at 10 mM Mg(II) without K (bullet) and with 0.1 M KCl (). D, the comparative activation of B. japonicum PBGS in bis-tris propane at neutral pH by KCl (bullet), NH(4)Cl (), and NaCl ().



A Monovalent Cation Effect on an Essential pK(a)

Preliminary characterization of the pH dependence of B. japonicum PBGS, illustrated in Fig. 3B, showed that the specific activity at neutral pH is highly dependent upon which buffer is used. This buffer effect was deduced to derive primarily from a variable monovalent cation concentration in different buffers or in any one buffer at different pH values. For example, Fig. 3C shows a pH activity curve in the buffer bis-tris propane-HCl, with 0 and 100 mM K added. The divalent cation Mg(II) was held at a saturating concentration of 10 mM (see below). The pH activity profile illustrates that the effect of K on B. japonicum PBGS is to lower a near neutral pK(a) by 1 pH unit. In the absence of potassium the apparent pK(a) is 7.3, and in the presence of potassium the apparent pK(a) is 6.4. Consequently, near neutral pH, K activation is dramatic. Both lines in Fig. 3C represent nonlinear least squares best fits for a single pK(a) ().

This simple one-proton pK(a) does not fit the experimental data well suggesting that there is more than one ionization involved. A more precise fit is not possible because many of the kinetic parameters vary as a function of pH (see below). A near neutral pK(a) has also been reported for type I and type II PBGS(20, 23, 24) .

The current study reveals that the effect of potassium on B. japonicum PBGS activity in the buffer bis-tris propane varies from as little as a 10% activation (1.1-fold) to as much as a 30-fold activation at pH values between 9 and 6 (Fig. 3C). Thus, at pH 6 potassium appears to be absolutely required for activity; at pH 7 potassium gives a 5-fold stimulation; and at pH values above 8 the effect of potassium is marginal. Several monovalent cations were tested as activators at neutral pH. Fig. 3D illustrates activation by K, Na, and NH(4) and shows that the apparent K(d) for NH(4) (0.5 ± 0.25 mM) < K (1.6 ± 0.9 mM) < Na (7 ± 4 mM), all with an equivalent V(max) of 38 µmol h mg. The data fit well to a single type of K binding site. Because K binding affinity may vary with pH, a 100 mM concentration of K was chosen for all subsequent studies that included K. For instance, at pH 6.5 the K(d) for K activation is found to be 14 mM.

Activity as a Function of Mg(II)

Characterization of types I and II PBGS established that this protein can use divalent metals ions for at least two, if not three, different functions (see Table 1and (7) ). The dissection of various functions for the same metal ion binding at various sites is not trivial. To distinguish the different roles of magnesium ions, Mg(II) activation of B. japonicum PBGS was investigated as a function of pH in the buffer bis-tris propane in the presence and absence of K. To ensure selection of appropriate assay conditions, K(m) and V(max) values were determined in bis-tris propane at several pH values in the presence and absence of K; the results are reported in Table 2. Fig. 4A illustrates the Mg(II) dependence of B. japonicum PBGS activity at pH 8.2, 6.9, and 6.5. In the presence of K (closed symbols), at all pH values studied, the Mg(II) activation curves can be fitted to a minimal two-site model where a tight binding Mg(II) (presumably Mg(A) or Mg(A) plus Mg(B)), is required for activity and a looser binding Mg(II) (Mg(C)) acts as an allosteric activator. Table 2shows the parameters of the best fit two-site model, which are represented by the lines in Fig. 4A. best describes these data, where K(d) is the dissociation constant for the required Mg(II), V(o) is the maximal activity in the absence of Mg(C), K(d) is the dissociation constant for Mg(C), and the activation factor is the -fold activation upon binding Mg(C).




Figure 4: The effect of Mg(II) and Zn(II) on B. japonicum PBGS activity. A, the Mg(II) dependence of B. japonicum PBGS activity as a function of pH and K in bis-tris propane. Closed symbols depict assays done in the presence of 100 mM KCl. Open symbols depict assays done without K. Almost no K was introduced with the protein. The pH values were 8.2 (up triangle, ); 6.9 (down triangle, ); and 6.5 (). B, correlation of activity () and the apparent K (circle) for ALA as a function of Mg(II) concentration in 100 mM KP(i) at pH 7.0. The assays were carried out in 0.1 M KP(i), pH 7, 10 µM Zn(II), 10 mM 2-mercaptoethanol, and contained 0.034 mg of enzyme. All assays containing 0.6 mM Mg(II) or less were allowed to proceed for 10 min before quenching. The apparent Kvalues for ALA were derived by the Lineweaver-Burke method (data not shown). C, B. japonicum PBGS specific activity as a function of total Zn(II) in the assay. All assays were done in 100 mM KP(i), pH 7, containing 10 mM 2-mercaptoethanol. For each curve, the concentration of Zn(II) was varied from 1 µM to 500 µM, and the concentration of Mg(II) was fixed at 1 µM (down triangle), 10 µM (up triangle), 100 µM (circle), 1 mM (), 3 mM (box), and 10 mM ().



The value of K(d), <0.3 µM at all pH values studied, is an upper limit because these assays used B. japonicum PBGS at 1 µM subunits; thus, total Mg(II) is a poor approximation for free Mg(II) at [Mg(II)] below 5 µM. V(o) is pH-dependent with values of 1.6, 5.0, and 22 µmol h mg at pH 6.5, 6.9, and 8.2, respectively (see Table 2). Consequently, although the maximum observed activity in the presence of K varies little with pH, the magnitude of activation by Mg(C) varies from 21-2-fold as the pH goes from 6.5 to 8.2.

In the absence of K (Fig. 4A, open symbols), the apparent K(d) for Mg(II) increases dramatically (Table 2), and kinetic evidence for ordered binding at two or more sites disappears. In this case the data fit to a cooperative model characterized by a maximal rate V`, a single K(d), and a Hill coefficient (n) of approximately 2 (see Table 2and ).

The data fit poorly to noncooperative single-site models. However, if Mg(C) can bind in the absence of the required Mg(II), the cooperativity apparent in the kinetic data need not indicate that Mg(II) binding is itself cooperative (see below). Some of the parameters listed in Table 2are approximations because the K(m) for ALA increases at subsaturating Mg(II). For instance, the reported V(max) for ALA at pH 6.9 in the absence of K does not represent true maximal velocities because Mg(II) is not fully saturating at 10 mM (see Table 2).

The Effect of Mg(II) on the K(m) for ALA

One well characterized effect of Mg(C) in type II PBGS is a reduction in the K(m) for ALA(9, 24) . Demonstration of a similar effect for B. japonicum PBGS would support our interpretation that the looser binding Mg(II) is indeed Mg(C). Fig. 4B shows an inverse relationship between Mg(II) activation of B. japonicum PBGS and the K(m) for ALA reminiscent of the Mg(C) effect on type II PBGS(9) . Here, in KP(i) buffer, the K(d) < 0.2 µM, V(o) = 3 ± 0.6 µmol h mg, K(d) = 0.7 ± 0.06 mM, and the activation factor = 10, similar to the parameters obtained in bis-tris propane with K at neutral pH. At Mg(II) concentrations approaching K(d) (e.g. 1 µM) the apparent K(m) for ALA approaches 1 M (data not shown).

Activity as a Function of Zn(II)

Fig. 4C shows that the effect of Zn(II) on the activity of B. japonicum PBGS is dependent upon the concentration of Mg(II). At neutral pH and saturating K the activity at low Zn(II) increases with increasing Mg(II) (see below). At very low Mg(II) (1 µM), the addition of Zn(II) activates slightly. At moderate levels of Mg(II) (100 µM), the addition of Zn(II) inhibits slightly. At high Mg(II), Zn(II) has no effect. In all cases where a Zn(II) effect is observed, the apparent binding constant for zinc is 200 µM, independent of Mg(II). A K(d) of 200 µM for Zn(II) is outside a reasonable physiologic range of free Zn(II). Therefore, these data suggest that Zn(II) can bind to sites normally occupied by Mg(II) with different functional consequences. At low Mg(II), Zn(II) binds to empty sites of B. japonicum PBGS and activates, although not as well as Mg(II). At moderate Mg(II), Zn(II) displaces Mg(II) from the activating site with a net inhibitory effect. At high Mg(II), Zn(II) cannot displace Mg(II) from the activating site, and no effect is observed. A Zn(II) binding determination carried out at 300 µM [Zn], 1 mMB. japonicum PBGS subunit and 1 mM Mg(II) revealed bound Zn(II) at 0.5/subunit (4/octamer). These high concentrations of both Zn(II) and protein define conditions under which the Zn(II) site (K(d) 200 µM) would be nearly saturated. Because the stoichiometry of the A-site is at 4/octamer and the A-site metal is required for activity, we deduce that Zn(II) binds at the A-site metal ion binding site. However, the high K(d) for Zn(II) suggests that Mg(A) is probably the physiologic metal ion.

Atomic Absorption Analysis of Bound Mg(II) and Zn(II)

To determine metal binding stoichiometries under various conditions, atomic absorption spectroscopy was utilized. The apparent K(d) of 0.6 mM for Mg(C) at pH 7 in 0.1 M KP(i) made it necessary to use high concentrations of protein to observe Mg(II) saturation of B. japonicum PBGS. Bound Zn(II) was also determined. B. japonicum PBGS, purified in the presence of 10 µM Zn, contains less than 0.1 Zn(II)/subunit. Mg(II) binding was determined at 1, 3, and 10 mM Mg(II) and revealed bound Mg at 0.32, 0.45, and 1.43/subunit, respectively (i.e. 2.6, 3.6, and 11.4/octamer). The Mg(II) stoichiometry at 10 mM Mg(II), where the kinetic data indicate saturation, reveals a total 1.5 Mg(II)/subunit or 12 Mg(II)/octamer. This is consistent with 4 Mg(A) and 8 Mg(C). The same stoichiometries were found in the presence of 5 mM porphobilinogen, which is thought to contribute one ligand to Mg(A).

The tight K(d) value for Mg(II) at pH 8.2 permits examination of metal binding by atomic absorption spectroscopy under assay conditions. Fig. 5illustrates the stoichiometry of bound Mg(II) as a function of Mg(II) concentration in TES-KOH and bis-tris propane (with 0.1 M K) at pH 8.2 in the presence and absence of ALA. In the absence of ALA, Mg(II) binding is dramatically reduced; the tight site is lost. Thus, the binding of the tight Mg(II) (Mg(A)) is dependent upon ALA, consistent with our model of A-side ALA as a bidentate ligand to the metal in site A. The data in the presence of ALA can be fitted to models in which B. japonicum PBGS binds Mg(A) at 0.5/subunit and Mg(C) at 1/subunit. The line represents this two-site model, where K(d) = <0.2 µM and K(d) = 40 µM. Fits to a single site are much worse. These data mirror those reported in Table 2. The line in the absence of ALA is consistent with a single site model where n = 1/subunit and K(d) = 120 µM.


Figure 5: Magnesium binding to B. japonicum PBGS in 0.1 M bis-tris propane-HCl at pH 8.2 containing 0.1 M KCl in the presence () or absence (up triangle) of 10 mM ALA and in 0.1 M TES-KOH in the presence () or absence (down triangle) of 10 mM ALA.



Magnesium binding stoichiometries were also determined using equilibrium dialysis and atomic absorption spectroscopy at 40 µM free Mg(II) under the five assay conditions illustrated in Fig. 4A in the presence and absence of ALA. The results, presented in Table 3, allow correlation of Mg(II) stoichiometries with the K(d) values determined from the kinetic studies. In the presence of K and ALA the values for bound Mg(II) illustrate a remarkable agreement with theoretical values calculated from the kinetic parameters of Table 2as long as one assumes that n = 0.5/subunit (4/octamer as for Mg(A)) and n = 1/subunit (8/octamer as for Mg(C)). For instance, at pH 8.2 the predicted value for bound Mg(II) at 40 µM free is 1.015/subunit, and the observed value is 0.994/subunit. On the other hand, in the absence of K (+ALA) if one presumes cooperative binding of Mg(A) and Mg(C), the experimental values of bound Mg(II) are much larger than the predicted values. Thus, in the absence of K, where no activity is observed at 40 µM Mg(II), Mg(C) must bind before Mg(A), and the experimentally determined values of bound Mg(II) can be used to estimate the K(d) of Mg(C) (see Table 3). Under all conditions studied, in the absence of ALA, the observed stoichiometry and affinity for bound Mg(II) is dramatically reduced. This result confirms that ALA enhances the binding of Mg(II). In the presence of K, the magnitude of the ALA enhancement is approximately equal to 0.5/subunit taking into account the K(d) values. K(d) values can also be calculated in the absence of ALA with and without K and are included in Table 3. ALA binding causes a 2-fold decrease in the K(d) for Mg(C) (K(d)). Similarly, K binding also causes a 2-fold decrease in K(d). In the absence of both, there is a 5-6-fold increase in K(d), showing a synergistic effect of K and ALA on Mg(C) binding. The effect of K and ALA on Mg(A) binding is a dramatic 10,000-fold decrease in K(d), from 1-2 mM to 0.2 µM. In the cases where ALA stimulates Mg(C) binding alone (e.g. without K), we presume ALA is binding at the P side ALA binding site. P side ALA is known to be the first ALA to bind to most PBGS at neutral pH(5, 25) .




DISCUSSION

Characterization of B. japonicum PBGS defines a new type IV PBGS, which complements information available about types I, II, and III PBGS. The following discussion places new knowledge about B. japonicum PBGS in the context of what is known about PBGS in general.

Protein Self-association Phenomena of PBGS

B. japonicum PBGS is the first PBGS to be documented to dissociate from the octamer to smaller active and inactive species under normal assay conditions. Gel filtration studies of Rhodobacter spheroides PBGS revealed dissociation from octamers to tetramers upon the removal of 50 mM potassium chloride(26) . In contrast, the dissociation of B. japonicum PBGS is observed at saturating (0.1 M) potassium. In an earlier study on R. spheroides PBGS, potassium was reported to cause the octamer to aggregate into species containing two and three octamers (27) . Prior work on type I (mammalian) PBGS showed an octameric structure that required denaturants to facilitate subunit dissociation (26) . Type II (E. coli) PBGS can be dissociated from octamers to hexamers, tetramers, and dimers by native gel electrophoresis, but dissociation is not observed by gel filtration (9) . Reaggregation of E. coli PBGS under assay conditions prevented comment on the intrinsic activity of species smaller than the octamer. The results of the current study are consistent with dimers, tetramers, and octamers all being active, while the monomer is not. This supports a growing body of evidence that the PBGS dimer is the fundamental unit of activity presumably because the active sites are at the dimer interfaces.

Monovalent Cation Interactions with PBGS

Almost all of the work of the past two decades on characterizing types I and II PBGS was carried out at K concentrations greater than 0.1 M following the precedent set by David Shemin (e.g. laboratories of Jordan, Beyersmann, Jaffe, Batlle and others). The earlier studies on the potassium effects on PBGS activity date from an era when the literature indicated that some PBGS (e.g. mammalian) do not require any metal ions or that monovalents could substitute for divalents in PBGS (for examples, see Refs. 3, 27). These misconceptions may have arisen from impurities present in commercial salts and buffers. Currently, monovalent and divalent metal ions are not generally thought to be interchangeable in their roles. This work on B. japonicum PBGS demonstrates that the monovalent cation effect is synergistic with, but not identical to, the divalent cation effects. In light of the pH dependence of the potassium effect on B. japonicum PBGS (see Fig. 3C), it is not possible to reevaluate the literature on other PBGS, as most studies were carried out at only one pH value. Recently, however, a pH-independent 2-fold stimulation of E. coli PBGS by potassium and phosphate was documented for a form of the enzyme containing only Zn(II)(28) .

The monovalent cation predominantly affects a pK(a) value on B. japonicum PBGS. The K(m) and V(max) data in Table 2show that in the presence of K, the pK(a) at 6.4 predominantly affects K(m) with marginal pH variation in V(max). This is similar to the K(m) and V(max) variations in the pK(a) of 7.5 of E. coli PBGS when it has its full complement of metal ions(24) . In the absence of K, both K(m) and V(max) are affected by pH.

Divalent Cation Interactions with PBGS

PBGS is now well documented to require at least one catalytic metal ion, which is generally accepted to interact with A-side ALA (e.g. the Zn(A) of type I PBGS)(7, 15, 29) . In the case of B. japonicum PBGS, this catalytic metal ion appears to be Mg(A). Because the coordination geometry of Zn(II) in proteins is usually tetrahedral or pentacoordinate and that of Mg(II) is uniformly octahedral (hexadentate), it is clear that the structure of the Zn(A)bulletA-side ALA complex will not be isosteric with the Mg(A)bulletA-side ALA complex. We believe that this is the first example of the natural evolution of a catalytic Mg(II) from (or to) a catalytic Zn(II). The data presented herein support an A-site metal ion stoichiometry of 4/octamer consistent with our prior studies and in contrast to the alternative interpretation of eight catalytic alpha-site metal ions bound to A-side ALA(15) .

Types II, III, and IV PBGS appear to share an allosteric metal ion, which in each case is filled by Mg(C)^3(9) . Only type I PBGS lacks Mg(C). To date no one has proposed a physiologic role for this allosteric metal ion. Since Mg(C) is the only Mg(II) in type II PBGS it follows that the effect of Mg(II) on type II PBGS must be due to Mg(C). Interpretation of the effect of Mg(II) on B. japonicum PBGS is less straightforward because B. japonicum PBGS binds both Mg(A) and Mg(C). An allosteric effect of Mg(C), reducing the K(m) for ALA binding to B. japonicum PBGS, mimicks that seen for type II PBGS(9, 24) . In addition, it is known that A-side ALA binding to type I and type II PBGS, at neutral pH, determines the apparent K(m) for ALA. Since A-side ALA binding is dependent upon a metal bound in site A (29) (which is apparently Mg(A) in B. japonicum PBGS), the Mg(II)-dependent decrease in K(m) must also be partially due to a Mg(A) requirement for A-side ALA binding.

The results presented herein are consistent with a model for active B. japonicum PBGS containing only Mg(A) and Mg(C), but not Mg(B). The B-site metal ion of type I PBGS, Zn(B), appears not to be essential for catalytic activity as long as its four cysteine ligands are protected from oxidation(12, 30) . Under normal physiologic conditions one role of Zn(B) is believed to be prevention of disulfide formation between sulfhydryl groups that participate in catalysis. It has been proposed that the role of these sulfhydryl groups is to accept a proton in porphobilinogen formation(11) . In types III and IV PBGS, aspartate could fill a similar role. In the case of type II PBGS, the cooperative binding of Zn(A), Zn(B), and Mg(C) has to date prevented definitive proof of whether or not Zn(B) is essential. The B. japonicum PBGS data again are consistent with the interpretation that the B-site divalent metal is nonessential.

What Determines a Preference for Zinc versus Magnesium?

Fig. 2illustrates a region of protein sequence first proposed to bind the required Zn(II) of type I PBGS(17) . Extended x-ray fine structure studies unequivocally established that the essential Zn(A) contains predominantly nonsulfur ligands and that Zn(B) uses four cysteines as ligands(13) . This is consistent with the generalization that Zn(II) ions with four cysteine ligands do not play a catalytic role(31, 32) , but raised the question of whether any of the Zn(A) ligands arise from the stretch of sequence illustrated in Fig. 2. Elegant cassette mutagenesis studies have now settled the question by placing this region of the human sequence into PBGS of pea^3 or B. japonicum to yield a hybrid protein whose activity responds to added Zn(II) while the parent protein does not(21) . Thus, at least some Zn(A) ligands arise from this region. The hybrid pea-human PBGS contained 1 Zn(II) and 1 Mg(II)/subunit, thus establishing that the Zn(B) ligands also reside in this region of the sequence.^3 The hybrid pea-human PBGS also unequivocally established that the allosteric Mg(C) binding site lies outside this region of sequence. The histidines of this region are possible Zn(A) ligands, but mutagenesis studies show that the HGH sequence does not form the nucleation site for Zn(A) binding(24) . Some of the ligands to Mg(A) of B. japonicum PBGS most likely arise from the aspartic acids of the region illustrated in Fig. 2; the ligands to Mg(B) are apparently not all present. The ligands to Mg(C) are yet to be determined.

The PBGS Reaction Mechanism

The PBGS-catalyzed reaction is a complex double dehydration reaction in which two identical substrate molecules condense asymmetrically (see Fig. 1). How this can occur at the dimer interface of two identical subunits is a puzzle that we hope to solve through the x-ray crystallographic structure of the protein (33) . Nevertheless, some aspects of the mechanism are well documented. First P side ALA binds and forms a Schiff base linkage at C-4 to an active site lysine (Lys in B. japonicum PBGS). We have recently established that B. japonicum PBGS utilizes the P-side Schiff base intermediate(6) . Next, A-side ALA binds as a (presumably bidentate) chelate of the divalent metal ion in site A. In keeping with this theme, here we show that binding of ALA facilitates the binding of Mg(A) at 0.5/octamer. The chemistry that follows formation of the ternary complex remains poorly described. The product porphobilinogen appears to contain a deprotonated amino group and binds quite tightly to the protein(34) . The pH activity curves revealed in this study all fit poorly to a single pK(a) and suggest that there are at least two protons in flight. This near neutral pK(a) has also been observed in type II PBGS where it was proven not to arise from the conserved histidines of PBGS(24) ; these are the only histidines in B. japonicum PBGS. Several deprotonations are required for porphobilinogen formation. Both ALA substrates, as well as the lysine that participates in the P-side Schiff base intermediate, are deprotonated at the amino group. Both C-3 protons of A-side ALA are lost during the reaction as is the pro-R proton at C-5 of P-side ALA. Any of these deprotonations could contribute to the observed pK(a).


FOOTNOTES

*
This research was supported by National Institutes of Health Grants ES03654 (to E. K. J.), CA06927 (to the Institute for Cancer Research), and CA09035 (to Fox Chase Cancer Center), and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3695; Fax: 215-728-2412.

(^1)
The abbreviations used are: PBGS, porphobilinogen synthase; ALA, 5-aminolevulinic acid; TES, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; bis-tris propane, 1,3-bis[tris(hydroxymethyl)- methylamino]propane.

(^2)
An alternative interpretation is that E. coli PBGS contains eight equivalent catalytically required Mg(A), which cannot bind in the absence of eight equivalent structural Zn(B)(15) .

(^3)
J. S. Ventura and M. P. Timko, personal communication.

(^4)
Under these conditions, the 2-mercaptoethanol is not required for activity.


ACKNOWLEDGEMENTS

We are grateful for the protein analytical chemistry carried out by Dr. William R. Abrams (University of Pennsylvania School of Dental Medicine and the mass spectral analysis carried out by Dr. David Andrews of ImmuLogic, Waltham, MA).


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