(Received for publication, June 5, 1995; and in revised form, August 30, 1995)
From the
Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by
glutamine. Significant changes in protein structure and activity were
observed in the H136Q and H140Q variants only. In contrast to wild-type
PPase, which is hexameric, these variants can be dissociated into
trimers by dilution, as shown by analytical ultracentrifugation and
cross-linking. Mg and substrate stabilize the
hexameric forms of both variants. The hexameric H136Q- and H140Q-PPases
have the same binding affinities for magnesium ion as wild-type, but
their hydrolytic activities under optimal conditions are, respectively,
225 and 110% of wild-type PPase, and their synthetic activities, 340
and 140%. The increased activity of hexameric H136Q-PPase results from
an increase in the rate constants governing most of the catalytic steps
in both directions. Dissociation of the hexameric H136Q and H140Q
variants into trimers does not affect the catalytic constants for
PP
hydrolysis between pH 6 and 9 but drastically decreases
their affinities for Mg
PP
and
Mg
. These results prove that His-136 and His-140 are
key residues in the dimer interface and show that hexamer formation
improves the substrate binding characteristics of the active site.
Phosphoryl transfer enzymes form one of the largest classes of
enzymes (Knowles, 1980), yet their mechanisms of action are still not
fully understood (Herschlag and Jencks, 1990). This class includes
soluble inorganic pyrophosphatases (EC 3.6.1.1; PPase), ()which hydrolyze inorganic pyrophosphate (PP
)
to inorganic phosphate (P
). These enzymes, essential in
both bacteria (Chen et al., 1990) and yeast (Lundin et
al., 1991), are ubiquitous and play an important role in energy
metabolism, providing a thermodynamic pull for biosynthetic reactions
such as protein, RNA, and DNA synthesis (Kornberg, 1962). According to
Peller(1976), nucleic acid synthesis would be energetically impossible in vivo if it were not coupled to the PP
hydrolysis catalyzed by PPases. The two best-studied soluble
PPases are those from the yeast Saccharomyces cerevisiae and Escherichia coli: each accelerates the rate of PP
hydrolysis by a factor of 10
compared with the rate
in solution. Detailed understanding of their catalytic mechanisms is
important for understanding the class of phosphoryl transfer enzymes as
a whole.
E. coli PPase is homohexameric (Wong et
al., 1970) and contains 175 amino acid residues per subunit (Lahti et al., 1988). Its three-dimensional structure, recently been
determined at 2.5-2.7-Å resolution (Kankare et
al., 1994; Oganessyan et al., 1994), is very like that of S. cerevisiae PPase (Kuranova et al., 1983; Terzyan et al., 1984), in accord with the conservation of active site
residues and mechanism in the two enzymes (Cooperman et al.,
1992; Kankare et al., 1994). E. coli PPase requires
four Mg ions per active site for catalysis, as
described in . This scheme, which fully accounts for the
overall catalysis of PP
:P
equilibration by E. coli PPase, is a slightly modified version of the one
proposed by Baykov et al.(1990) following the approach
developed for S. cerevisiae PPase (Springs et al.,
1981; Welsh et al., 1983).
The cloning and sequencing of the E. coli ppa gene that encodes PPase (Lahti et al., 1988) together with the analysis of the conservation of functional residues between yeast and E. coli PPase (Lahti et al., 1990a) have made it possible to study the structural and functional relationship of E. coli PPase by site-directed mutagenesis (Lahti et al., 1990b). All 17 polar residues located in the active site cavity have been substituted (Lahti et al., 1990b; Lahti et al., 1991; Cooperman et al., 1992) and some of the variant PPases have been characterized in detail (Salminen et al., 1995; Käpyläet al., 1995).
Earlier chemical modification studies of Samejima et al. (1988) had implicated histidines as being involved in the activity of E. coli PPase. The absence of His residues from the active site cavity (Kankare et al., 1994) and the lack of conserved His residues in soluble PPases (Cooperman et al., 1992) make it clear that His residues have no direct role in catalysis, leaving open the possibility that the chemical modification results arise from an indirect effect. In this work we demonstrate that substitution of each of the five His residues in E. coli PPase with Gln results in substantial retention or even enhancement of catalytic activity, confirming their nonessential character. However, in contrast to wild-type enzyme, two variants, H136Q and H140Q, are found to dissociate readily into trimers, providing valuable insight into the details of subunit:subunit interaction.
The hexameric and trimeric forms of H136Q- and H140Q-PPases were
obtained by varying the composition of their stock solutions in order
to shift the hexamer &rlhar2; trimer equilibrium in the desired
direction (see ``Results''). Stock solutions of hexameric
PPase contained 225-1000 µM enzyme, 1 mM (for
H136Q) or 50 mM (for H140Q) MgCl, and 0.15 M Tris-HCl, pH 7.2. Dilutions were made with the same buffer
containing 20 mM (for H136Q) or 50 mM (for H140Q)
MgCl
. Because the H140Q-PPase dissociated so rapidly, we
had to add 0.3 mM PP
to the dilution medium for
this enzyme and could keep the diluted solution for no longer than 15
s, during which PP
was still present. The stock solution of
trimeric H136Q-PPase contained 0.9 µM enzyme, 1 mM MgCl
, and 0.15 M Tris-HCl, pH 8.5; that of
trimeric H140Q-PPase contained 2.25 µM enzyme, 1 mM MgCl
, and 0.15 M Tris-HCl, pH 7.2. All enzyme
solutions contained, in addition, 50 µM EGTA, and 0.5
mg/ml bovine serum albumin was added to the dilution media. All stock
solutions were preincubated for at least 30 min before use.
where k is the catalytic constant for
synthesis (see ``Results''). Rates of oxygen exchange between
P
and H
O were measured by mass spectrometry as
described by Baykov et al.(1990). Other kinetic and binding
measurements as well as calculations of various rate and binding
constants were carried out as described by
Käpyläet al.(1995).
Unless otherwise indicated, the media used were buffered with Tris-HCl,
the concentration of which was varied to maintain the ionic strength at
0.15-0.20 M. EGTA (typically 50 µM) was
included in all solutions containing enzyme. All experiments reported
were carried out at 25 °C.
Figure 2:
Specific activities of H136Q-PPase (A) and H140Q-PPase (B) pre-equilibrated at different
enzyme and Mg concentrations and pH.
, pH 7.2,
50 mM Mg
;
, pH 7.2, 1 mM Mg
;
, pH 8.5, 1 mM Mg
. The incubation media contained 1 mg/ml
bovine serum albumin. Following the incubation, enzyme activity was
assayed at pH 7.2 in the presence of 20 µM
Mg
PP
and 20 mM Mg
.
The lines are drawn according to , using parameter values given
in Table 1.
These results were analyzed in terms of :
using the following relationships (Kurganov, 1982):
where A is the observed specific activity, A and A
are the specific
activities of trimeric and hexameric enzyme, respectively;
is the fraction of hexameric enzyme at equilibrium
(
=
6[H]/([E]
);
[E]
is total enzyme concentration in
terms of monomer. Fitting these equations to the data (Fig. 2)
yielded the K
values shown (Table 1). When
fitting the H140Q data obtained at 1 mM Mg
,
the value of A
was fixed at 191
s
, the limiting value of activity at infinite enzyme
concentration as determined from the profile obtained at 50 mM Mg
for this enzyme.
As seen from Fig. 2and Table 1, increasing the pH destabilizes the
H136Q variants but stabilizes the H140Q variant in the presence of 1
mM Mg. Measurements of the equilibrium
activity at a fixed enzyme conentration (0.11 µM)
indicated that pH 7.2 is optimal for the stability of H136Q-PPase while
the stability of H140Q-PPase increases monotonically up to pH 10.1
(data not shown).
Figure 1: Cross-linking of wild-type and variant PPases by glutaraldehyde. Wild-type (2), H136Q (4), and H140Q (6) PPases in 4-30% gradient polyacrylamide gel in the presence of 0.2% sodium dodecyl sulfate following cross-linking with glutaraldehyde. Lanes 1 (wild-type), 3 (H136Q), and 5 (H140Q) show the mobility of non-cross-linked samples on SDS-polyacrylamide gel electrophoresis. The positions of the molecular mass markers (in kDa) are indicated (Lane S). The gels were stained with Coomassie Blue R-250.
Other data are also consistent with
the existence of a hexamer-trimer equilibrium in these two variant
PPases and support the assumption made above that the effects of enzyme
and Mg concentrations and pH on their specific
activities result from shifts in this equilibrium. Firstly, the
sedimentation coefficient (s
) changes
with changing pH and Mg
concentration (Table 2), and this effect correlates with the activity data (Fig. 2). It should be noted that for H140Q-PPase, the
sedimentation profiles were generally broader than for H136Q-PPase,
suggesting that both hexamers and trimers are present in significant,
although not equal, amounts. Accordingly, most s
values are somewhat lower for
hexamer and greater for trimer versus H136Q-PPase (Table 2). Secondly, analyzing the electrophoretic mobilities of
the H136Q and H140Q variants as functions of polyacrylamide
concentration (pH 9.5, in the absence of Mg
) (Hedrick
and Smith, 1968) indicated a 2-fold decrease in their molecular weights
compared to that of WT-PPase (data not shown). By contrast, a D97E
variant, whose specific activity is independent of enzyme
concentration, does not show such a decrease
(Käpyläet al., 1995).
Figure 3:
Formation of enzyme-bound PP by H136Q and H140Q-PPases in the presence of 20 mM free
Mg
. Enzyme concentration was 100-140
µM. The PP
concentration of acid-quenched
samples was determined either from
P radioactivity
(Springs et al., 1981) (
,
) or using a coupled
enzyme assay (Baykov et al., 1990; Nyren and Lundin, 1985)
(
).
, H136Q, pH 7.2;
, H136Q, pH 8.0;
, H140Q,
pH 7.2. The lines are drawn according to using best-fit
values for the parameters (Table 3).
The results of oxygen exchange measurements at two saturating
Mg concentrations (Table 4) were used to
calculate the catalytic constant for exchange k
and the partition coefficient P
= k
/(k
+ k
) (Hackney and Boyer, 1978). The k
values were calculated by extrapolating v
to infinite [MgP
] with , using the K
, K
,
and K
values in Table 3. Finally, Fig. 4shows the effects of Mg
concentration on
the synthesis and liberation of PP
to the medium (medium
PP
synthesis). This is the only activity of E. coli PPase inhibited by excess Mg
(Baykov et
al., 1990). The asymptotic values of v
at 20
mM Mg
and above were assumed to be the
corresponding catalytic constants k
. From the
above parameters (Table 3), we calculated the rate constants k
-k
in (Table 5). Special care was taken to ensure that the
variant PPases were hexameric during these measurements (see
``Experimental Procedures''). Additionally, hydrolytic
activity was measured at 20 µM Mg
PP
(20 mM Mg
, pH 7.2) to verify that the
hexameric structure is retained by the end of enzyme incubation with
P
and Mg
in the oxygen exchange and
enzyme-bound PP
measurements.
Figure 4:
Rates of PP synthesis to
medium by wild-type and variant PPases as functions of
Mg
concentration. MgP
concentration was
fixed at 20 mM (
,
,
) or 10 mM (
). Rates were measured by a coupled enzyme assay (Baykov
and Shestakov, 1992).
, wild-type PPase, pH 7.2;
,
H136Q-PPase, pH 7.2;
, H136Q-PPase, pH 8.0;
,
H140Q-PPase, pH 7.2.
pH-rate studies (see
below) showed that the maximal activity of the H136Q variant,
approximately twice its activity at pH 7.2, occurs at pH 8.0-8.5.
Therefore, we also determined the rate constants k-k
for this variant at
pH 8.0. As for WT-PPase, (
)its metal binding affinities at
pH 8.0 were greater than at pH 7.2. The metal binding constants could
not, however, be determined accurately because the H136Q variant
dissociates rapidly at pH 8.0 and above if the concentration of metal
ion and/or substrate is low, as is required for such an analysis. The
values of most, if not all, rate constants for H136Q-PPase are higher
than those of WT-PPase at pH 8.0 (Table 5).
Three lines of evidence rule out the possibility
that the activities measured result from hexamer formation during the
assay. First, hexamer formation is a slow reaction. For H136Q variant,
the association rate constant is only 0.77
µM min
at pH 7.2
(see below), which corresponds to half-time of 9 days for hexamer
formation at 0.1 nM enzyme concentration, which we routinely
use in hydrolysis assays. Second, the product formation curves are
strictly linear during 3 min of the assay, indicating that no
interconversion between enzyme forms occurs during the assay. Third,
the measured K
values are very different from
those for the hexameric forms.
Figure 5:
Dependence of k (A) and k
/K
(B) on pH for H136Q-PPase. The parameters were measured
for hexameric H136Q-PPase (circles) and trimeric H136Q-PPase (triangles) in the presence of 20 mM (open
symbols) or 50 mM (closed symbols)
Mg
. The lines represent best fits of and , taking into consideration all data in A and only data obtained at 50 mM Mg
in B.
The results at 50 mM Mg
were used to calculate apparent ionization constants for enzyme with
substrate bound, indicated as ESH, and for enzyme lacking
substrate, indicated as EH, as well as of pH-independent
values for k
and
k
/K
at 50 mM Mg
, according to and .
Parameter values are presented in Table 6, along with
those determined from similar data (not shown) for the H140Q hexamer
(we were unable to determine pH-rate profiles for H140Q trimer). For
the most part, values for the hexameric variants are quite similar to
those for wild-type. The only notable differences are the 2-fold
increase in k for H136Q variant (see below) and
the increases that both variants show in the values of
pK
and
pK
. Mutations of active site
residues also give rise increases in pK
and pK
, but the magnitude of
such increases is considerably greater (Salminen et al.,
1995). The H136Q trimer shows a still larger increase in
pK
as well as a large decrease in k
/K
.
Fitting and , in combination with and , to the data shown in Fig. 6A
generated k values shown in Fig. 6B.
K
values paralleled with k
,
indicating that the effect of metal concentration on k
is small. The dissociation constant for Mg
of
2.4 mM was obtained by fitting k
to :
Figure 6:
Protection of H136Q-PPase by
Mg against inactivation on dilution. A, time
courses of the apparent inactivation of H136Q-PPase upon dilution in
the presence of 0.04 (
), 0.5 (
), 2 (
), 5 (
), 10
(
), and 20 (
) mM Mg
. Stock
enzyme solution containing 50 µM enzyme, 20 mM MgCl
, 1 mM EGTA, 1 mM
dithiothreitol, and 0.2 M Tris-HCl, pH 7.2, was diluted to 0.1
µM with the same medium containing different amounts of
Mg
, incubated for the indicated time intervals at 25
°C and assayed for residual activity at pH 7.2 with 20
µM Mg
PP
and 20 mM Mg
. The curves represent the best fit for and . B, dissociation rate constant,
as calculated from A, versus Mg
concentration. The line is drawn according to using K
= 2.4 mM and k
= 0.18
min
.
where k,
is k
at zero Mg
concentration and K
is the metal binding constant.
A similar analysis was carried
out for the H140Q variant (data not shown). For this variant k was as high as 0.6
s
, and the data were more scattered. Nevertheless,
the value of K
we obtained, 1.8 mM, was
again close to the K
values derived above from
hydrolysis kinetics and equilibrium dialysis measurements (Table 3). Despite the high rate constant for hexameric H140Q
dissociation, the P
liberation curves in the assay of
residual activity were linear for at least 3 min, indicating that
substrate profoundly stabilizes the hexameric form.
Figure 7: MOLSCRIPT (Kraulis, 1991) stereo schematics of wild-type E. coli PPase. A, a monomer of E. coli PPase. Helices are represented as spirals and sheets as curved arrows. The side chains of His-136 and His-140 are shown in a ball-and-stick representation, as are the positions of some key active site residues: Asp-70, Lys-29, Arg-43, and Lys-142. B, the interface between two monomers around the noncrystallographic 2-fold axis in wild-type E. coli PPase. Helix A is shown as a spiral, and His-136, His-140, Lys-142, and Asp-143 are numbered, and their side chains are represented by ball-and-stick models. One monomer has primed (`) numbers, the other, not. The x in the center of the figure marks the position of the noncrystallographic two-fold axis, and the network of hydrogen bonds is shown with dashed lines.
It seems very
likely that, per monomer at neutral pH, a single positive charge is
shared between His-136 and His-140. (The resolution of the current
structure is not sufficient to tell which is the more likely His.)
Consequently, replacing either His will either weaken or destroy the
ion pair depicted in Fig. 7B. In both cases, the
replacement Gln can orient to allow its -amido group to replace
the lost His-Asp hydrogen bond, thus partially mitigating the effect of
the substitution. The helix-forming tendency of glutamine is slightly
higher than that of histidine (O'Neil and DeGrado, 1990), thus
making unlikely any destabilization of the
-helix A in the variant
proteins.
Based on the structure of the subunit contact region, one
would expect, and it is indeed observed (Table 1), that the
hexameric structure of the H136Q variant is more stable than that of
the H140Q variant. His-136 is more exposed to solvent than His-140 so
the Gln-136 side chain in the H136Q variant can swing out of the way to
allow solvation both of Asp-143` and of Gln-136. Conversely, in the
H140Q variant, the completely buried Gln140 will have unsatisfied
hydrogen bond donors and acceptors that cannot be solvated (at least
one His on the -amido group and lone pairs on the
-carbonyl
group). In addition, there is a loss of hydrophobic interaction between
His-140 and His-140` in the H140Q variant. Finally, because 140 is the
most buried residue, the conformation of Gln-140 will be much more
constrained than the conformation of Gln-136 in the two respective
variants: as a result, more entropy will be lost on forming H140Q
hexamers than on forming H136Q hexamers.
Another clear difference
between the two variants is in their pH-stability profiles: the H136Q
variant shows a pH optimum at about pH 7.2. This is consistent with two
Asp:HisH
ion pairs stabilizing
subunit association, with the loss of stability at lower and higher pH
being due to protonation of Asp-143 and deprotonation of His-140,
respectively. Here we note the report of Borshchik et
al.(1986) that even WT-PPase will dissociate into trimers on
incubation at pH 5. At present we have no cogent rationale for the pH
stability profile of the H140Q variant.
Measured at pH 8.0, close to the optimum
for both enzymes, k and k
are 2-3-fold greater for the H136Q variant than for
wild-type PPase, as are most of the microscopic rate constants (Table 5). At least three possible explanations can be suggested
for why this is so. First, the mutation may somehow optimize active
site conformation. Second, weakening intersubunit interactions may
increase conformational flexibility of the protein molecule, which may
be important provided that some reaction steps involve rate-limiting
conformational changes. Finally, the trimers in WT-PPase may interact
such that the perfect hexameric symmetry breaks down and one trimer is
always inactive, while in the H136Q-PPase the trimers are equivalent
and active. Alternatively, the trimers could cycle: one
``on'' and one ``off'' as, for example, proposed
for ATP synthase (Boyer, 1993; Abrahams et al., 1994).
Hexameric H136Q-PPase would thus be more active than (hexameric)
WT-PPase because its trimers would be more independent. We cannot, at
present, select between the above possibilities. It is, however,
intriguing that E. coli PPase crystallizes in two different
forms. One has obligate perfect identity between all six monomers
because the space group is R32 and there is only one monomer in the
asymmetric unit (Heikinheimo et al., 1995). The other has two
monomers in the asymmetric unit of a double-sized R32 unit cell and so
the trimers, related by a noncrystallographic 2-fold axis (Kankare et al., 1994), are not constrained to be identical and,
indeed, appear not to be. (
)
In terms of the
three-dimensional structure, the binding changes are explained by the
fact that the interactions discussed above (Fig. 7B)
not only stabilize the monomer-monomer interface but also define a
specific conformation for the 141-143 loop that immediately
follows helix A. That loop contains one of the more important active
site residues. Mutation of Lys-142 to Arg-142 results in a large
increase in the K for Mg
PP
(Salminen et al., 1995) and in weakened binding for both
MgP
and a competitive inhibitor of
Mg
PP
. (
)Consequently, any
destabilization of the 141-143 loop on hexamer dissociation might
be expected to affect the active site and, in particular, the binding
of PP
and P
(Salminen et al., 1995).
The greater affinity of hexameric PPase for Mg
and
Mg
PP
explains why these ligands stabilize it versus trimeric PPase.
We demonstrated (data not shown)
that the increased K is an intrinsic property of
the trimers, as opposed to arising from an indirect effect of each
mutation on the active site, by showing that WT-PPase trimer, formed by
prolonged incubation at low pH following Borshchik et
al.(1986), resembles the variants studied in this paper in having
a k
value similar to that of the hexamer but much
higher K
values. This latter result differs from
that reported by Borshchik et al.(1986), who claimed that the K
values of trimer and hexamer were similar. It
is, however, consistent with reports of activity loss following
dissociation of the structurally related PPases of the thermophilic
bacteria PS-3 (Hachimori et al., 1979) and Bacillus
stearothermophilus (Schreier, 1980) into trimers. Our work
suggests that this loss of activity is due to an effect on K
. Finally, by analogy with our present results,
we speculate that the loss of E. coli PPase activity found by
Samejima et al.(1988) on chemical modification of His residues
arises from dissociation of hexamer into trimers.