(Received for publication, November 8, 1995; and in revised form, January 30, 1996)
From the
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 present at a stoichiometry of 4/octamer, an
allosteric Mg
present at a stoichiometry of 8/octamer, and
a monovalent metal ion, K
. However, the divalent
Mg
or Zn
present in some other PBGS is not
present in B. japonicum PBGS. Under optimal conditions, the K
for Mg
is <0.2
µM, and the K
for Mg
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. However, consistent
with prior predictions, B. japonicum PBGS can bind Zn(II)
(presumably as Zn
) at a stoichiometry of 4/octamer with a K
of 200 µM; but this high
concentration is outside a physiologically significant range.
Porphobilinogen synthase (PBGS, ()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, decreases
the K
for ALA, and decreases the K
for 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
and four
Zn
. E. coli-like PBGS is type II; it contains four
Zn
, four Zn
, and eight Mg
. (
)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) ; (
)it hypothetically contains four
Mg
, four Mg
, and eight Mg
, 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) . 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.
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, 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
, pH
7.0, 12 mM MgCl
, 40 µM ZnCl
, 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
buffer at pH 7.0 containing 113 g/liter ammonium sulfate (20%
saturation, loading buffer) and 1 mM MgCl
, 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
buffer at pH 7.5 containing 1 mM MgCl
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
buffer at pH 7.5 containing 1 mM MgCl
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.
where S is a subunit and S 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
is the concentration of n multimers
present.
Expressing C as a function of N and the K
values yields the following.
Newton's method was used to calculate N as a function of C(22) . The specific activity
(SA) was calculated as follows, where
is a theoretical V
.
These equations allowed us to determine specific activities for
each concentration C and any set of constants K, K
, K
, and
. The space of these four constants
was searched to fit the calculated specific activities to the observed
values by the least squares method.
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 caused by monovalent cations.
These properties are described first because they dictate the
conditions used for the divalent cation studies.
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 (). 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 (- - -),(- - -), (-
-), and (-
-) 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 (
,
); 0.1 M TES-KOH (
,
); 0.1 M KP
(
,
). 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
(
) and with 0.1 M KCl
(
). D, the comparative activation of B. japonicum PBGS in bis-tris propane at neutral pH by KCl (
),
NH
Cl (
), and NaCl
(
).
This simple one-proton pK 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
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
and shows that the apparent K
for NH
(0.5
± 0.25 mM) < K
(1.6 ± 0.9
mM) < Na
(7 ± 4 mM), all
with an equivalent V
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
for K
activation is found to be
14 mM.
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 (
,
); 6.9 (
,
); and 6.5 (
). B, correlation of activity (
) and the apparent K
(
) for ALA as a function of Mg(II)
concentration in 100 mM KP
at pH 7.0. The assays
were carried out in 0.1 M KP
, 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 K
values 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
, 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 (
), 10 µM (
), 100 µM (
), 1 mM (
), 3 mM (
), and 10
mM (
).
The value of K, <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
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
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
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
, and a Hill coefficient (n) of approximately 2 (see Table 2and ).
The data fit poorly to noncooperative single-site models.
However, if Mg 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
for ALA increases at subsaturating Mg(II). For
instance, the reported V
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 tight K 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
) 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
at 0.5/subunit and Mg
at
1/subunit. The line represents this two-site model, where K
= <0.2 µM and K
= 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
= 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 (
) of 10
mM ALA and in 0.1 M TES-KOH in the presence (
)
or absence (
) 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
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
) and n
= 1/subunit (8/octamer as for
Mg
). 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
and Mg
, 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
must bind before Mg
, and the
experimentally determined values of bound Mg(II) can be used to
estimate the K
of Mg
(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
values. K
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
for
Mg
(K
). Similarly,
K
binding also causes a
2-fold decrease in K
. In the absence of both, there
is a 5-6-fold increase in K
,
showing a synergistic effect of K
and ALA on Mg
binding. The effect of K
and ALA on Mg
binding is a dramatic 10,000-fold decrease in K
, from
1-2 mM to
0.2 µM. In the cases where ALA stimulates
Mg
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) .
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.
The monovalent cation predominantly affects a
pK value on B. japonicum PBGS. The K
and V
data in Table 2show that in the presence of K
, the
pK
at 6.4 predominantly affects K
with marginal pH variation in V
. This is
similar to the K
and V
variations in the pK
of 7.5 of E. coli PBGS when it has its full complement of metal ions(24) .
In the absence of K
, both K
and V
are affected by pH.
Types II, III, and IV PBGS appear to share an allosteric metal ion,
which in each case is filled by Mg(9) .
Only type I PBGS lacks Mg
. To date no one has proposed a
physiologic role for this allosteric metal ion. Since Mg
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
. Interpretation of the
effect of Mg(II) on B. japonicum PBGS is less straightforward
because B. japonicum PBGS binds both Mg
and
Mg
. An allosteric effect of Mg
, reducing the K
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
for
ALA. Since A-side ALA binding is dependent upon a metal bound in site A (29) (which is apparently Mg
in B. japonicum PBGS), the Mg(II)-dependent decrease in K
must also be partially due to a Mg
requirement for
A-side ALA binding.
The results presented herein are consistent with
a model for active B. japonicum PBGS containing only Mg and Mg
, but not Mg
. The B-site metal ion
of type I PBGS, Zn
, 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
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
,
Zn
, and Mg
has to date prevented definitive
proof of whether or not Zn
is essential. The B.
japonicum PBGS data again are consistent with the interpretation
that the B-site divalent metal is nonessential.