(Received for publication, March 7, 1995; and in revised form, July 19, 1995)
From the
Porphobilinogen synthase (PBGS) is a metalloenzyme that
catalyzes the first common step of tetrapyrrole biosynthesis, the
asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA)
to form porphobilinogen. Chemical modification data implicate histidine
as a catalytic residue of PBGS from both plants and mammals. Histidine
may participate in the abstraction of two non-ionizable protons from
each substrate molecule at the active site. Only one histidine is
species-invariant among 17 known sequences of PBGS which have high
overall sequence similarity. In Escherichia coli PBGS, this
histidine is His. We performed site-directed mutagenesis
on His
, replacing it with alanine. The mutant protein
H128A is catalytically active. His
is part of a
histidine- and cysteine-rich region of the sequence that is implicated
in metal binding. The apparent K
for
Zn(II) binding to H128A is about an order of magnitude higher than for
the wild type protein. E. coli PBGS also contains
His
which is conserved through the mammalian, fungal, and
some bacterial PBGS. We mutated His
to alanine, and both
His
and His
simultaneously to alanine. All
mutant proteins are catalytically competent; the V
values for H128A (44 units/mg), H126A (75 units/mg), and
H126/128A (61 units/mg) were similar to wild type PBGS (50 units/mg) in
the presence of saturating concentrations of metal ions. The apparent K
for Zn(II) of H126A and H126/128A is
not appreciably different from wild type. The activity of wild type and
mutant proteins are all stimulated by an allosteric Mg(II); the mutant
proteins all have a reduced affinity for Mg(II). We observe a
pK
of
7.5 in the wild type PBGS k
/K
pH profile as
well as in those of H128A and H126/128A, suggesting that this
pK
is not the result of
protonation/deprotonation of one of these histidines. H128A and
H126/128A have a significantly increased K
value for the substrate ALA. This is consistent with a role
for one or both of these histidines as a ligand to the required Zn(II)
of E. coli PBGS, which is known to participate in substrate
binding. Past chemical modification may have inactivated the PBGS by
blocking Zn(II) and ALA binding. In addition, the decreased K
for E. coli PBGS at basic pH
allows for the quantitation of active sites at four per octamer.
Porphobilinogen synthase (PBGS) ()(EC 4.2.1.24)
catalyzes the asymmetric condensation of two molecules of ALA to form
porphobilinogen. This reaction is the first common step in the
tetrapyrrole biosynthetic pathway, which is responsible for the
formation of porphyrins, chlorins, corrins, and other essential
cofactors(1) . There is high sequence similarity among the 17
documented sequences of PBGS from a phylogenetically diverse selection
of prokaryotes and
eukaryotes(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 20) .
A review of data on the PBGS mechanism has been published
recently(21) . All PBGS are metalloenzymes, and there is an
intriguing phylogenetic variation in the use of metal ions.
Under
physiologic conditions, Escherichia coli PBGS contains Zn(II)
and Mg(II), each at a stoichiometry of eight per octamer (or two per
active site)(22, 23) . The Zn(II) are believed to be
analogous to the eight Zn(II) of mammalian PBGS; four Zn participate in catalysis and four Zn
appear to bind
essential cysteine residues(24, 25, 26) .
Zn
and Zn
are both at the active
site(27) . For E. coli PBGS, it is difficult to
differentiate between the two types of bound Zn(II), and
Zn
Zn
is used to denote enzyme-bound Zn(II) in
both sites of E. coli PBGS. The eight equivalent Mg(II)
(termed Mg
) act as allosteric activators and have no
counterpart in the mammalian protein(23) , but appear to be
common to bacterial and plant PBGS(21, 28) . Although
not at the active site, Mg
causes a 50-fold increase in k
/K
, affecting
both parameters favorably. Mg
increases the affinity of the
protein for Zn
Zn
(23) , but Mg
cannot bind to PBGS unless Zn(II) is bound(22) .
It
has been proposed that both Zn and Zn
draw
ligands from a putative metal binding domain in a mutually exclusive
fashion and that Mg
binds elsewhere in the
sequence(21, 25, 26, 28) . Fig. 1shows the sequence of the proposed metal binding domain.
The technique of extended x-ray absorption fine structure spectroscopy
discriminated between Zn
and Zn
on the basis of
the chemical nature of their ligands (26) . The conserved
tyrosine, aspartate, and histidines of the metal binding domain are
candidates for some of the predominantly non-sulfur ligands to a
pentacoordinate Zn
. The four cysteines of this region are
proposed to tetrahedrally coordinate Zn
. In those plant and
bacterial species where the cysteines are not present, there are many
aspartic acid residues, and Mg
is proposed to functionally
replace Zn
(17, 21) .
Figure 1:
The putative metal binding region of
PBGS. The region from amino acid 110 to 135 in E. coli PBGS is
shown along with the homologous region from 16 other sequences.
Proposed Zn(II) ligands are in boldface. The four cysteines
are proposed to be ligands to Zn, whereas the tyrosine,
aspartate, and/or histidine(s) of this region are potential ligands to
Zn
(see text). The residues mutated in this study are underlined in the E. coli sequence, and the
substitutions are illustrated directly above these residues in parentheses. The numerous aspartates in PBGS of eukaryotic
photosynthetic organisms are proposed to bind
Mg
(11) .
The PBGS-catalyzed
reaction (shown in Fig. 2) involves the overall loss of two
water molecules. Various proposed mechanisms all include the
abstraction of one or more protons from the substrate or an
intermediate by a general base (e.g.(29, 30, 31) ). This general base may be
histidine; published pH rate profiles show a near-neutral
pK, which is consistent with a
catalytically essential histidine (28) and
diethylpyrocarbonate, a group-specific reagent which targets histidine,
inactivates both porcine and maize PBGS(32, 33) .
His
(numbered as in the E. coli sequence and
shown in Fig. 1) is the only species invariant histidine in the
PBGS sequence and thus is an attractive candidate for an essential
histidine.
Figure 2: The PBGS-catalyzed reaction. The asymmetric condensation of two molecules of ALA involves removal of four non-ionizable hydrogen atoms and the two C-4 carbonyl oxygen atoms. A-side ALA loses both C-3 methylene protons, and P-side ALA loses one proton from the C-5 methylene group and one from the amine group. The detailed chemical mechanism remains a matter for speculation, but it is likely to involve acid-base chemistry at the active site(21) .
Histidine at the active site of PBGS might also serve as
a Zn(II) ligand. Extended x-ray absorption fine structure spectroscopy
studies of bovine PBGS indicate that Zn (which binds
directly to the substrate(25) ) contains at least one imidazole
ligand(26) . Diethylpyrocarbonate inactivation of porcine PBGS
can be protected against by the addition of Zn(II), suggesting that the
modified histidine is a Zn(II) ligand(32) . His
in the E. coli sequence has previously been proposed to
be a ligand to Zn
(25) . His
and
His
may form the nucleation site for binding
Zn
. Recently His
was proposed to be the key
``switch'' in determining a preference for binding Zn(II)
(mammals and some bacteria) or Mg(II) (plants and some bacteria) to the
region illustrated in Fig. 1. (
)
To investigate
whether His or His
are involved in
catalysis and/or Zn
ligation, we performed site-directed
mutagenesis on E. coli PBGS to replace His
and
His
with alanine, both in turn and simultaneously.
Alanine was selected because the side chain methyl group of alanine is
unable to serve either as a general base or as a Zn(II) ligand. A small
side chain was chosen to have a minimal effect on protein folding. We
report here the kinetic and other physical properties of the mutant
proteins relative to wild type E. coli PBGS. These results
also provide significant new information about wild type E. coli PBGS.
Figure 3:
The effect of metal ions on the activity
of wild type and mutant PBGS. A, the effect of added Zn(II)
with no added Mg(II). , wild type;
, H126A;
, H128A;
, H126/128A. Filled symbols represent assays done in the
presence of 1 mM Mg(II). Assays were performed as described
under ``Materials and Methods''; intrinsic [Zn(II)]
is
1 µM. B, response of activity to added
Mg(II) at 100 µM Zn(II).
, wild type;
,
H126A;
, H128A;
, H126/128A. The left-most symbols represent Zn(II)
1 µM and Mg(II)
1
µM for graphs A and B,
respectively.
We previously
established that Mg is an allosteric activator of wild type
PBGS which increases the V
, reduces the K
for ALA, stabilizes the quaternary structure,
and reduces the K
for
Zn
Zn
(23) . The multiple functions of
Mg
are retained in the mutant proteins but the magnitudes
of the effects vary. The kinetic parameters for mutant and wild type
PBGS (determined at 10 µM Zn(II), ± 1 mM
Mg(II)
) are shown in Table 3. Mg
causes V
to increase by 3.4-13.2-fold and K
to decrease by 5-40-fold for the mutant
proteins. Native polyacrylamide gel electrophoresis reveals similar
stabilization of the quaternary structure of the mutant proteins by
Mg
. Fig. 3B shows the effect of added
Mg(II) on the activity of wild type and mutant PBGS and can be used to
estimate the K
for Mg
. H126A, H128A,
and H126/128A all have a higher K
for Mg
(0.4, 0.3, and 0.5 mM, respectively) than wild type PBGS
(<0.1 mM).
Figure 4:
The effect of pH on the kinetic parameters
of wild type PBGS, H128A, and H126/128A. , wild type;
,
H128A;
, H126/128A. A, k
/K
(M
s
) versus pH; B, K
(mM) versus pH (note y axis is log scale); C, V
(units/mg) versus pH. These
experiments were performed as described under ``Materials and
Methods'' for the determination of kinetic constants. The pH shown
is measured after the addition of 10 mM ALA
HCl to the
assay mix. Assays also contained 10 mM
ME, 10 µM Zn(II), and 1 mM Mg(II).
Figure 5:
[3,5-C]Porphobilinogen
binding to E. coli PBGS (3 mM subunits) saturates
four active sites/octamer. Integrated signal intensities from the C-3
resonance of free porphobilinogen (
, expressed in mM)
and bound porphobilinogen (
, expressed as bound/octamer) were
quantified.
Figure 6:
Arrhenius plots of wild type PBGS and
H126/128A. , wild type;
, H126/128A. k
is calculated as the turnover number. Each point is the average
of four determinations. Lines are least squares linear fit.
Assays included a 10-min preincubation at the indicated
temperature.
Site-directed mutagenesis has been used to probe the roles of
one species-invariant histidine His of E. coli PBGS and its partially conserved neighbor His
.
Previous chemical modification data had suggested an essential
histidine(32, 33) . The most striking result is the
retained ability of the mutant proteins H126A, H128A, and H126/128A to
catalyze the formation of porphobilinogen, indicating that these
histidines are not essential for catalysis. Under approximately
physiologic conditions the V
and K
values for the mutant proteins vary little from
wild type E. coli PBGS, suggesting that the rate-determining
step(s) of the reaction are unaltered by the mutation. Thus, one may
ask why these histidines are so highly conserved. Perhaps these
residues are involved in a step in catalysis whose rate is several
orders of magnitude faster than the rate-determining step; the overall
rate of PBGS is slow (k
is
1
s
). The PBGS-catalyzed reaction includes several
essential steps. P-side ALA (see Fig. 2) must bind and form a
Schiff base to Lys
. Zn
must bind to
facilitate binding of A-side ALA. Beyond catalyzing formation of these
early complexes, the protein may play a passive role, simply providing
the scaffolding for binding substrates and metal ions. The conserved
residues may contribute only secondary interactions to catalysis.
Mutation of His and/or His
to alanine
perturbs the K
for ALA; the K
is determined by the more loosely bound of the two identical
substrates, the ALA known to require Zn
in order to bind at
the active site(25, 38) . This increased K
is consistent with our active site model because
the increase may be due to altered affinity for Zn
or to
alteration of the A-side ALA binding pocket. Despite the value of K
for the mutant proteins, they maintain the
pK
of 7.5 in the k
/K
profile. This
pK
cannot be due to His
nor
His
; the possibility that this pK
arises from another histidine is diminished by the fact that some
PBGS (e.g.Bradyrhizobium japonicum PBGS) contain
only these two histidines. Since this pK
must be
related to substrate binding, it may help verify one of two published
models for A-side ALA binding to Zn
, shown in Fig. 7. One model shows Zn
bound through the
carbonyl oxygen of ALA (Fig. 7A)(39) , whereas
an earlier model shows bidentate ligation through both the amino group
and the carbonyl oxygen (Fig. 7B)(25, 40) . The bidentate
model arises in part from
C and
N NMR spectra
of the protein-product complex which showed the amino group of bound
porphobilinogen to be deprotonated(40) . The pK
of 7.5 may reflect deprotonation of the amino group of
enzyme-bound ALA which would be necessary for bidentate chelation.
However the pK
for deprotonation of the amino
group of free ALA is 8.4.
Figure 7:
Proposed models for ALA bound to
Zn. A, ALA chelated through the C-4 carbonyl
oxygen only(37) ; B, ALA chelated through both the C-4
carbonyl oxygen and the C-5 amino
nitrogen(28, 39) .
His and/or His
have been introduced as potential Zn(II) ligands. A review of the
literature indicates that the mutation of Zn(II) ligands can have
different effects on different proteins. For bacteriophage T4 gene 32
protein, mutation of the Zn(II) binding residues Cys
or
His
result in a mutant protein which does not bind Zn(II)
or single-stranded DNA and shows some structural instability in the
Zn(II) binding region(41) . However, mutation to a Zn(II)
ligand of mammalian sorbitol dehydrogenase (42) or E. coli methionyl-tRNA synthetase (43) results in a reduced
affinity for Zn(II) which can be overcome by adding excess Zn(II) to
the assay. This is like the H128A mutant of PBGS. It appears that the
removal of one ligand of four or five may be insufficient to eliminate
metal binding because Zn(II) can accommodate a variable number of
ligands equally well. Recent ab initio calculations of Bock et al.(19) show little energetic differences among
complexes with Zn(II) bound to four, five, or six first coordination
sphere water molecules. Although removal of one or two ligands may not
disrupt Zn(II) binding, simultaneous mutation of two neighboring
ligands may have a dramatic effect. Metalloproteins often contain a
nucleation site for metal ion binding composed of two amino acids
separated by three or fewer intervening amino acids in the primary
structure(15) . In E. coli PBGS, His
and
His
comprise one possible Zn(II) nucleation site. The
affinity of H126/128A for Zn(II) indicates that these two histidines do
not form the nucleation site for Zn(II) binding.
PBGS from some
species have been reported to require Mg(II) but not Zn(II) (e.g.(17) ). Our current results raise the question whether the
Mg effect can be so large as to mask a requirement for
Zn(II). For instance, Mg
has a 21-fold effect on V/K of wild type PBGS and a 134-fold effect for
H126A. Thus, a relatively minor change in the PBGS sequence can
dramatically affect the magnitude of the allosteric effect of
Mg
. In addition, Mg
enhances the binding
affinity for Zn
Zn
, amplifying the apparent
allosteric effect at low Zn(II). Thus, care must be taken in evaluating
the metal ion requirements for PBGS from all species.