 |
INTRODUCTION |
Inorganic pyrophosphatase (EC 3.6.1.1;
PPase)1 catalyzes
specifically interconversion of pyrophosphate and orthophosphate. Owing
to the simplicity of its substrate, PPase is a convenient system to
study the mechanism of phosphoryl transfer from polyphosphates, including nucleoside triphosphates, to water, an essential but still
incompletely understood biochemical transformation. Soluble PPase is
essential for life (1, 2), since it provides a thermodynamic pull for
biosynthetic reactions (3).
Until recently, only one family (family I) of soluble PPases had been
known, of which the PPases of Saccharomyces cerevisiae and
Escherichia coli are the most extensively characterized
representatives (4, 5). Despite variability of subunit size and of
quaternary structure (eukaryotic PPases are dimers of 30-35 kDa
subunits, whereas prokaryotic PPases are hexamers of ~ 20 kDa
subunits), PPases of family I have a highly conserved active site
structure formed by 14-16 amino acid residues and 3-4
Mg2+ ions and very similar catalytic properties. Catalysis
by these enzymes proceeds via direct attack of water on a phosphorus
atom without formation of a covalent intermediate. The metal ions are the key to catalysis and mediate the major protein-PPi
interactions, which serve to shield the charge on the electrophilic
phosphorus, activate the nucleophilic water molecule, and increase the
acidity of the leaving phosphate group (6-8).
Recently, a long-known PPase of Bacillus subtilis (9) was
found to have a completely different amino acid sequence and therefore
to belong to a different family (family II) of soluble PPases (10, 11).
B. subtilis PPase is similar to family I PPases in requiring
divalent metal ions for activity (9), but unlike bacterial family I
PPases, it is formed by large subunits (34 kDa) and displays 10-20
times greater activity (9, 12). Another unique property of B. subtilis PPase is its preference for Mn2+ over
Mg2+ as the activator. A search through
GenBankTM revealed four more putative prokaryotic members
of family II (two streptococcal and two archeal), showing 40-57%
identity in amino acid sequence (10, 11). One of them (from
Methanococcus jannaschii) has been recently cloned and
expressed in E. coli (13). The catalytic mechanism employed
by family II PPases and the structural basis for their remarkable
activity remain to be elucidated.
In this work, we cloned and overexpressed in E. coli the
putative genes for family II PPases from Streptococcus
mutans, implicated together with dietary sugars as the principal
cause in the development of dental caries (14), and Streptococcus
gordonii, another human oral bacterium. The recombinant proteins
were purified and shown, along with the B. subtilis PPase,
to be highly active dimeric PPases with a unique requirement for
Mn2+.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, and Other
Materials--
Restriction endonucleases were purchased from Fermentas
(Vilnius, Lithuania), and Dynazyme polymerase was purchased from
Finnzymes (Espoo, Finland). The plasmid vector pET-15b was
obtained from Novagen (Madison, WI), and primers for
polymerase chain reaction (PCR) were from Medprobe (Oslo, Norway). The
QuikChange site-directed mutagenesis kit was purchased from Stratagene
(La Jolla, CA), and a Wizard DNA Clean-up system was from Promega
(Madison, WI). DEAE fast flow, phenyl-Sepharose CL-4B and Superdex 200 prep grade columns were from Amersham Pharmacia Biotech.
E. coli XL2blueb (Stratagene) and E. coli C43(DE3) (15) were used as hosts in the cloning and
expression, respectively. S. gordonii ATCC 10558 and
S. mutans ATCC 25175 were obtained from the Culture
Collection of the University of Göteborg (Sweden) and the
Institute of Dentistry (University of Turku, Finland), respectively.
The E. coli strains were grown in 2× YT broth or on
LA plates (16). Ampicillin (100 µg/ml) was added when required.
DNA Manipulation and Protein Expression--
Genes for Sg-PPase
and Sm-PPase were expressed in E. coli under the inducible
phage T7 promoter by making use of the pET system. Chromosomal DNA was
isolated from both streptococcal strains as described by Ushiro
et al. (17). The open reading frames encoding Sg-PPase and
Sm-PPase were amplified by PCR using a 5'-sense oligonucleotide primer
containing a restriction site for NcoI and a 3'-reverse complement primer with a BamHI site. The PCR product was
purified by using the Wizard DNA Clean-up system, digested with
NcoI and BamHI, and ligated into the vector
pET-15b. In creating the NcoI site, a T
G mutation after
the initiation codon (ATG) was made both in S. gordonii and
S. mutans PPase genes. After ligating the PCR products into
the vector, the mutations were reversed by using the QuikChange
site-directed mutagenesis kit, and the produced DNA constructions were
transformed into E. coli XL2blueb and E. coli C43(DE3) for DNA sequencing and expression, respectively. The
expression was induced for 5-6 h by 1 mM
isopropyl-1-thio-
-D-galactopyranoside. B-PPase was
expressed in E. coli as described by Shintani et
al. (11).
Protein Purification--
Sg-PPase and Sm-PPase were purified to
homogeneity by DEAE fast flow ion exchange column chromatography in 20 mM Tris/HCl, pH 7.3, 10 mM MgCl2,
10 mM MnCl2 followed by gel filtration on Superdex 200 prep grade in 0.15 M Tris/HCl, pH 7.2, 15 mM MgCl2, 1.5 mM MnCl2.
B-PPase was additionally chromatographed on a phenyl-Sepharose column
that was equilibrated with 1.7 M
(NH4)2SO4 in the buffer used for
the ion exchange chromatography and eluted with a downward gradient of
(NH4)2SO4 concentration. Enzyme
purity was checked by electrophoresis in 8-25% gradient
polyacrylamide gels in the presence of 0.55% SDS using the Phast
System (Amersham Pharmacia Biotech).
Metal ions were removed from the enzyme stocks (30-50 mg/ml) as
follows. The enzyme solution was diluted 20-fold with 83 mM TES/KOH buffer, pH 7.2, containing 2 mM EDTA and 17 mM KCl, incubated for 2 days (B-PPase) or 4 days (Sg-PPase
and Sm-PPase) at 4 °C and subjected to two 40-fold
dilution/reconcentration cycles in a Centricon YM-30 centrifugal filter
device (Amicon) using 83 mM TES/KOH buffer, pH 7.2, containing 50 µM EGTA and 17 mM KCl. Stock
PPase solutions and other solutions used in experiments involving long
incubations were sterilized by passing them through a 0.2-µm filter.
Concentrations of B-PPase, Sg-PPase, and Sm-PPase were determined on
the basis of extinction coefficients

of 0.264, 0.343, and 0.307, calculated from the amino acid composition using the program
ProtParam and the subunit molecular masses of 34.0, 33.5 and
33.4 kDa, respectively, calculated from the amino acid sequences (11).
The above value of 
for
B-PPase was confirmed by direct measurement of the absorbance of the
solution prepared from dried and weighed enzyme. The Bradford method
(18), standardized against the above method, was sometimes employed as
an alternative to direct measurement of the absorbance.
Activity--
Rates of PPi hydrolysis were
determined from continuous recordings of Pi liberation
obtained using an automatic Pi analyzer (19). Reactions
were initiated by adding enzyme.
Sedimentation--
Analytical ultracentrifugation was carried
out in a Spinco E instrument (Beckman) with scanning at 280 nm.
Sedimentation velocity was measured at 48,000-60,000 rpm, and the
sedimentation coefficient, s20,w, was calculated
using a standard procedure (20). Sedimentation equilibrium was attained
at 16,000 rpm for 16 h or at 24,000 rpm for 10 h, and the
molecular mass was calculated according to Chernyak and Magretova (21).
The partial specific volume at 25 °C was calculated from the amino
acid composition and found to be 0.735, 0.729, and 0.730 cm3/g for B-PPase, Sg-PPase, and Sm-PPase, respectively.
Equilibrium Dialysis--
Mg2+ and Mn2+
binding was assayed by equilibrium microdialysis in combination with
atomic absorption spectroscopy to measure Mg2+ and
Mn2+ content in the dialysis chambers (22).
Except where noted, all activity and binding measurements were
performed at 25 °C in the medium containing 83 mM
TES/KOH buffer, pH 7.2, 50 µM EGTA, and 17 mM
KCl. For incubations with Mg2+, EGTA concentrations were
increased to 0.5 mM. Bovine serum albumin was added at a
concentration of 1 mg/ml to all incubation media, except for those used
in sedimentation and equilibrium dialysis.
Calculations--
Equations 1 and 2, derived from Scheme I,
describe the activity (A) of an equilibrium mixture of dimer
(D) and monomer (M) as a function of enzyme concentration at a zero or
fixed concentration of divalent metal ion. AD
and AM are the specific activities of dimer and
monomer, respectively, [E]t is the total enzyme concentration, expressed in monomers,
D is the fraction
of dimeric enzyme, kd and
ka are the rate constants, and
Kd = kd/ka is the dissociation
constant.
|
(Eq. 1)
|
|
(Eq. 2)
|
The Mn2+ concentration dependence of the equilibrium
activity could be described by Equations 1 and 2 in combination with
Equations 3-5, where AM,
AD', and AD" are the
activities of monomer, metal-free dimer, and metal-bound dimer,
respectively, Kd,0 is Kd at zero
Mn2+ concentration, and KM1 is the
dissociation constant governing Mn2+ binding to dimer.
Equation 5 is an implicit extended mass balance equation for metal. The
second and third terms on the right side of Equation 5 correspond to
protein-bound and EGTA-bound metal, respectively, [EGTA]t is
the total concentration of EGTA in the system (50 µM),
and KMEGTA is the dissociation constant for its
complex with Mn2+. This treatment implies that only dimer
can bind Mn2+, which has a dual effect on
activity;Mn2+ both increases the amount of dimer (by
decreasing Kd) and further activates it (by
increasing Ad). Equations 1 and 2 or 1-5 were
simultaneously fit to data with the program SCIENTIST (MicroMath).
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
The dissociation constants for Mn-EDTA and Mn-EGTA complexes at
pH 7.2 (0.013 and 6.3 nM, respectively) used to estimate
the concentrations of free Mn2+ in solutions containing
EGTA and EDTA were calculated from the stability constants of their
deprotonated Mn2+ complexes taking into account the
pKa values of EGTA and EDTA (23).
 |
RESULTS |
Cloning and Expression of the Streptococcal PPase Genes--
The
putative open reading frames of Sg-PPase and Sm-PPase were amplified by
PCR. By sequencing the genes we noticed Sg-PPase to have four
differences from the gene-deduced amino acid sequences found from
GenBankTM; positions 109, 135, 137, and 166 are occupied by
Ser, Gly, Pro, and Val (the corresponding codons are AGT, GGC, CCA, and
GTC), respectively, rather than by Asn, Ser, Ser, and Ala, as found in
GenBankTM. These differences were reproduced in two
independent PCR amplifications, ruling out possible mutations during
gene manipulations.
The PCR products expressed under phage T7 promoter in E. coli yielded transformants with about 100-fold higher PPase
activity than the host strain, clearly indicating that the two open
reading frames encode PPases. Because of the high expression level, the recombinant enzymes were easily purified to homogeneity by ion exchange
and gel filtration chromatography, the first of these steps readily
separating the chromosome-encoded E. coli PPase from the
streptococcal PPases. From 50 to 100 mg of pure enzymes were obtained
from 1 liter of cell culture, corresponding to about 5 g of cell paste.
Quaternary Structure--
The sedimentation coefficients measured
for the B. subtilis and the two streptococcal PPases in the
presence of Mg2+ or Mn2+ by the sedimentation
velocity method were within 3.5-4.1 S (Table I). In the absence of the divalent metal
ions (2 mM EDTA present), s20,w
decreased markedly for B-PPase and less markedly for Sg-PPase and
Sm-PPase, indicating dissociation into lower molecular mass species.
View this table:
[in this window]
[in a new window]
|
Table I
Sedimentation coefficients
Before each run, the enzymes were preincubated for 1-3 h (B-PPase) or
3 days (Sg-PPase and Sm-PPase) in the buffer containing the indicated
additions. The s20,w values are precise to ±0.2 S.
|
|
The sedimentation equilibrium method gave molecular masses of 63 ± 3 kDa for B-PPase and 68 ± 5 kDa for Sg-PPase in the presence of 1.5 mM MnCl2 (Fig.
1) and 32 ± 2 kDa for B-PPase in
the presence of 2 mM EDTA. The molecular masses of B-PPase
and Sg-PPase dimers predicted from their amino acid sequences (11) are
68.0 and 67.1 kDa, respectively. These proteins are therefore clearly
dimers in the presence of MnCl2, but B-PPase dissociates
into monomers in the absence of the metal ions. The assumption of a
monomeric or trimeric structure in the presence of MnCl2
resulted in a poor fit (Fig. 1).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Sedimentation equilibrium distribution of
Sg-PPase in the presence of 1.5 mM MnCl2 at
25 °C. The relative concentration of Sg-PPase measured by its
absorbance at 280 nm was plotted as a function of the radial distance
at 16,000 rpm. The initial enzyme concentration was 20 µM. The experimental points were fitted to a homogeneous
species model with a single molecular mass of 33.5 kDa (dotted
line), 67.1 kDa (solid line), or 100.6 kDa
(dashed line). The residuals shown on the top are for the
best fit, obtained with the 67.1-kDa molecular mass.
|
|
Activity Versus Enzyme Concentration Profiles--
The equilibrium
between different oligomeric forms of enzyme can be studied by
measuring its specific activity as a function enzyme concentration if
the specific activities of the oligomeric forms differ and their
interconversion is slow on the time scale of enzyme assay (24).
Earlier, we used this approach in studies of E. coli PPase
variants with weakened quaternary structure (25-28). The specific
activities of the three PPases under study increased with increasing
enzyme concentration in the stock solution containing 2 mM
EDTA (no free divalent metal ion) or 1.5 mM
Mg2+ and remained unchanged if the stock solution contained
1.5 mM Mn2+ (Fig.
2). The activity values shown, except for
the Sm-PPase-EDTA curve (see below), correspond to equilibrium,
as indicated by comparison with similar curves obtained after 1 and 2 days of incubation (not shown; see also Fig. 4 below). Three sources of evidence indicated that the profiles for B-PPase and Sg-PPase incubated
with EDTA and the profiles for all three PPases incubated with
Mg2+ in Fig. 2 describe a monomer-dimer equilibrium. First,
they obeyed Equations 1 and 2 derived for such an equilibrium. Second,
the enzymes preincubated at low concentration could be completely reactivated upon the addition of 3.5 mM MnCl2,
as illustrated in Fig. 3 for Sg-PPase.
Moreover, the reactivation rate increased with increasing enzyme
concentration, exhibiting half-times of 14 and 1.4 min at enzyme
concentrations of 1.8 and 18 nM, respectively, as expected
for the second-order reaction of dimer formation from monomers. By
contrast, the half-times would be similar for a first-order reaction,
involving only one molecule of the reactant. Third, these findings
agree with the sedimentation data above, showing that
s20,w is greater in the presence of
Mn2+ or Mg2+ than in the presence of EDTA at
20-26 µM enzyme concentration (Table I).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Specific activities of family II PPase
pre-equilibrated at different enzyme concentrations. The enzymes
were preincubated for 1 day (B-PPase) or 3 days (Sg-PPase and Sm-PPase)
at 25 °C with 2 mM EDTA (closed circles), 1.5 mM MgCl2 (open circles), or 1.5 mM MnCl2 (triangles), and their
activities were assayed with 57 µM PPi, 20 mM MgCl2, 40 µM EGTA, and 0.15 M Tris/HCl, pH 7.2. The lines are drawn
according to Equations 1 and 2 using the parameter values shown in
Table II.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Time courses of activity during
Mn2+-stimulated dimerization of Sg-PPase at 1.8 nM (open circles) or 18 nM
(closed circles) enzyme concentration. Sg-PPase
pre-incubated at 0.1 µM concentrations with 2 mM EDTA as in Fig. 2 was diluted with the buffer
supplemented with 3.5 mM MnCl2. Aliquots were
withdrawn in time and assayed for PPase activity as in Fig. 2. The
lines are drawn according to second-order kinetics using the
ka values given in the text.
|
|
The activity of Sm-PPase preincubated with EDTA was only partially
restored upon the addition of MnCl2, indicating
irreversible inactivation during the incubation. Interestingly, the
inactivation was significant only at [E]t > 50 µM and increased with increasing [E]t,
suggesting the occurrence of enzyme aggregation. This explanation is
supported by the observation that Sm-PPase, but not B-PPase or
Sg-PPase, could be precipitated by heating its solution above
30 °C.
Three immediate inferences can be made from the data in Figs. 2 and 3
and the parameter values listed in Table
II. First, dimers are more active than
monomers. Second, Mg2+ and, especially, Mn2+
stabilize dimer versus monomer (compare the
Kd values), primarily due to a change in
ka. Dimer stability is the lowest with B-PPase.
Third, the AD values are low and similar in
magnitude with Mg2+ and EDTA (at least for B-PPase and
Sg-PPase) but high with Mn2+, indicating that
Mn2+ activates dimer, whereas Mg2+ does
not.
View this table:
[in this window]
[in a new window]
|
Table II
Parameters for monomer-dimer equilibrium
Parameter values were calculated from the dependencies shown in Figs.
2-4, except for the values of kd in the presence of
Mn2+ and values of ka in the presence of
EDTA and Mg2+, which were calculated as
kaKd and
kd/Kd, respectively.
|
|
Metal-dependent Activity Modulation--
Fig.
4 shows the time courses of activity upon
dilution of the EDTA-treated PPases into media containing EDTA,
Mg2+, or Mn2+. In all cases EDTA stimulated
inactivation, and Mn2+ stimulated activation, whereas the
effect of Mg2+ was variable and much smaller. The
inactivation by Mg2+ and EDTA proceeded on the time scale
of hours, whereas the activation by Mn2+ was complete in
30 s, except for a small slower phase seen with Sm-PPase.
Importantly, the enzymes were predominantly dimeric at the start of
incubation (Fig. 2); therefore the major activation is not due to
stimulation of dimer formation, except for the slower phase seen with
Sm-PPase (Fig. 4). By contrast, the inactivation clearly results from
dimer dissociation into monomers because the data in Fig. 2 indicated
that the PPases equilibrated with EDTA or Mg2+ at the
concentrations used for Fig. 4 represent a mixture of dimer and
monomer. A similar pattern of inactivation by EDTA was reported for
wild-type B-PPase (12). Values for kd (Scheme I) estimated from the data in Fig. 4 are summarized in Table II.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Time courses of PPase activity during
incubation with Mg2+ (open circles),
Mn2+ (triangles), or EDTA (closed
circles). For the incubation with Mn2+,
stock solution of B-PPase (2.4 mM) was diluted to 26 µM using the buffer supplemented with 1.5 mM
MnCl2, and aliquots were withdrawn over time for activity
assay as in Fig. 2. In all other cases, stock solutions of B-PPase,
Sg-PPase, and Sm-PPase (2.4, 3.2, and 1.1 mM enzyme
concentrations, respectively) were diluted to 20 µM with
the buffer supplemented with 50 µM EGTA and then further
diluted within 2 min to 0.1 µM with the buffer
supplemented with 1.5 mM MgCl2, 1.5 mM MnCl2, or 2 mM EDTA. The
lines for the EDTA and Mg2+ incubations are
drawn for a first-order reaction with the kd
values shown in Table II.
|
|
The Mn2+ concentration dependence of the equilibrium
activity (Fig. 5) could be described by
assuming that Mn2+ selectively binds to dimer (with a
dissociation constant of KM1), thus shifting the
equilibrium in Scheme I in the direction of dimer, and further
activates it. Estimates of KM1 (Table
III) were obtained by fitting Equations
1-5 to the data in Fig. 5. Values of AD" and
KM1 were treated as adjustable parameters in
these fittings, and values of AM,
AD'. and Kd,0 were
constrained to the values of AM,
AD, and Kd determined above
in the presence of EDTA (Table II). For Sm-PPase, no
Kd,0 and AD' values are
available (see above); therefore, Kd,0 was
set to zero, and AD' was also treated as an
adjustable parameter, allowing estimation of the upper limit for
KM1. Remarkably, the values of
KM1 estimated for all three PPases are extremely
low (Table III), characteristic of metalloenzymes.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Mn2+ concentration dependence of
PPase activation. The enzymes (20-26 µM) were
pre-equilibrated with Mn2+ for 1 day (B-PPase) or 4 days
(Sg-PPase and Sm-PPase) before activity was assayed as in Fig. 2. The
upper scale refers to free Mn2+ concentration in the medium
before PPase was added and, therefore, neglects Mn2+
binding by PPase. The lines are drawn according to Equations
1-5 using the AM, Kd, and
KM1 values found in Tables II and III (see
"Results").
|
|
Direct Measurements of Mn2+ and Mg2+
Binding--
As measured by equilibrium dialysis, dimeric Sg-PPase has
one high affinity and two or three low affinity sites for
Mn2+ and Mg2+ per subunit (Fig.
6). The binding curves for both cations
exhibited saturation, not clearly seen in Fig. 6 because of the
logarithmic scaling of the metal concentration axis. The high affinity
site demonstrated a marked preference for Mn2+ over
Mg2+, whereas the low affinity sites bound Mn2+
with only slightly greater strength than they bound Mg2+.
Very similar binding curves were obtained for B-PPase and Sm-PPase (not
shown). The dissociation constant KM1,obs
characterizing metal binding to the high affinity site was estimated
from these data using only points measured at <1 µM for
Mn2+ and <100 µM for Mg2+ (Table
III). The values of KM1,obs thus obtained did
not vary much between the three PPases, and the values of
KM1,obs for Mn2+ binding to B-PPase
and Sg-PPase were slightly greater than the corresponding values of
KM1 determined above. This difference appears to
result from the presence of appreciable amounts of monomer, which lacks
the high affinity site possessed by the dimer, at low metal ion
concentrations. For the same reason, values of KM1,obs for Mg2+ shown in Table III
may also exceed the corresponding KM1 values for
this cation.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Mn2+ and Mg2+ binding
to Sg-PPase as measured by equilibrium dialysis. n is
the number of metal ions bound per subunit, and the abscissa
shows free metal ion concentrations. The incubation medium contained
410-800 µM PPase, 10-3000 µM
Mg2+ or 1-3500 µM Mn2+ and 50 µM EGTA (total concentrations). The equilibration was
performed for 4 days at 25 °C. The solid lines are drawn
for a one-site model using the KM1,obs values
shown in Table III.
|
|
In the presence of 50 µM Mn2+, no high
affinity site was seen for Mg2+, and the total binding
stoichiometry decreased by one in Sg-PPase (Fig.
7) and B-PPase (not shown).This is
consistent with Mn2+ and Mg2+ competing for the
same M1 site, which binds Mn2+ more tightly than
Mg2+ by 4 orders of magnitude (Table III). Mg2+
binding to the low affinity sites changed insignificantly in the
presence of 50 µM Mn2+ (Fig. 7).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Mn2+ and Mg2+ binding
to Sg-PPase as measured by equilibrium dialysis at fixed free
Mn2+ concentration (50 µM). PPase concentration was
700-900 µM.
|
|
Metal Ion Requirements for Activity--
No hydrolytic activity
was observed when the family II PPases were assayed in the absence of
divalent metal ions. The activity was maximal when Mn2+ was
present in the preincubation or assay medium or in both of these media
(Fig. 8, curves Mn/Mn,Mg,
Mg/Mn,Mg, Mn/Mn, Mn/Mg). However, if
Mn2+ was present only during preincubation, the activity
gradually decreased during the assay, resulting in a nonlinear
Pi production curve (curve Mn/Mg). The latter
effect clearly resulted from displacement of Mn2+ from the
high affinity site by Mg2+. That the curve
Mg/Mn,Mg was linear and had the maximal slope suggested that
the back substitution occurs quite rapidly during catalysis.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
Time courses of PPi hydrolysis by
Sg-PPase. The enzyme was pre-equilibrated for 3 days with 1.5 mM Mn2+ or 1.5 mM Mg2+
and assayed with 57 µM PPi in the presence of
1 mM Mg2+, 1 mM Mg2+
plus 0.1 mM Mn2+, 0.1 mM
Mn2+, 0.5 mM Ca2+, or no added
metal ion. Labels, metal ion in the preincubation/metal
ion(s) in the assay. The enzyme concentration in the preincubation mix
was 0.045 µM (Mn) or 0.9 µM
(Mg) and in the assay mix was 0.015 nM
(Mn/Mn,Mg, Mg/Mn,Mg, Mn/Mn,
Mn/Mg, Mg/Mg) or 0.11 nM
(Mn/Ca, Mn/none).
|
|
The Michaelis-Menten parameters for the Mg2+-activated
PPi hydrolysis were determined for dimeric and monomeric
PPases obtained by preincubations identical to those used for Fig. 2.
The preincubations with Mn2+ or Mg2+ at the
enzyme concentrations indicated in Table
IV yielded dimer, whereas the
preincubations with EDTA (no divalent metal ion present) yielded a
mixture of monomer and dimer, with monomer being the dominant species.
Fitting of rate versus substrate concentration profiles (not
shown) to the sum of two Michaelis-Menten equations, one for monomer
and the other for dimer, yielded kcat and
Km values for monomers and Km
values for dimers. The results presented in Table IV indicate that
preincubation with Mn2+ renders dimeric PPases better
catalysts in terms of kcat and worse catalysts
in terms of Km than preincubation with Mg2+ or without divalent metal ions.
View this table:
[in this window]
[in a new window]
|
Table IV
Michaelis-Menten parameters for dimer and monomer in
Mg2+-activated PPi hydrolysis
The enzymes were preincubated as for Fig. 2 and assayed in the presence
of 20 mM Mg2+. Values of kcat
for dimeric enzymes preincubated without metal ions were calculated
from the AD values given in Table II and the
Km values shown here (see "Results" for
details). Values of Km are in terms of total
PPi concentration.
|
|
Ca2+ is known to strongly inhibit family I PPases by
replacing the activating Mg2+ in its complex with enzyme
and substrate (29). Typically, activity drops by 50% at
[Ca2+]/[Mg2+]
1/50. By contrast, no
inhibition (
15%) was observed when the family II PPases were assayed
in the presence of 2-10 mM Ca2+ (20 mM Mg2+, 57 µM total
PPi). Moreover, Ca2+ was able to activate
family II PPases preincubated with Mn2+ (compare curves
Mn/Ca and Mn/none in Fig. 8). Although the
activities measured with 0.5 mM Ca2+ (64 s
1 with B-PPase, 135 s
1 with Sg-PPase, and 160 s
1 with Sm-PPase) were only about 10% of the
activities conferred by Mn2+, they compare well with the
activities of family I PPases measured with their best activator,
Mg2+. However, Ca2+ did not support
PPi hydrolysis by Sg-PPase and Sm-PPase, from which
Mn2+ was removed by preincubation with EDTA
(activities less than 0.2 and 0.004 s
1, respectively).
Substrate Specificity--
Dimeric PPases preincubated with 1.5 mM Mn2+ and assayed in the presence of 0.17-2
mM Mn2+ or 20 mM Mg2+
plus 0.07 mM Mn2+ exhibited low but measurable
activity against tripolyphosphate (0.03-0.06
s
1 at 57 µM substrate) but not
against ATP (<0.004 s
1) (data not shown).
 |
DISCUSSION |
Family II of Soluble PPases--
A new family of soluble PPases
(family II) has recently been observed by two research groups (10, 11).
The first verified member of family II PPases was B. subtilis PPase, but in addition, amino acid sequences of four
putative members of this family were found in the
GenBankTM, two streptococcal and two archeal (11). The
results shown above indicate that the two streptococcal sequences do
belong to highly efficient and specific PPases. Furthermore, the open reading frames encoding the putative family II PPases of M. jannaschii (13) and Archaeoglobus
fulgidus2 have recently
been expressed in E. coli and also shown to be PPases.
Table V summarizes the major properties
distinguishing families I and II of the bacterial PPases. Kinetically,
family II PPases are superior in terms of kcat,
but their full activity can hardly be manifested in vivo
because of high Km values. PPi strongly
inhibits the biosynthesis pathways, which produce it as a by-product,
and for this reason it is unlikely to be accumulated to the levels
comparable with Km. As a result of high Km, the hydrolysis rate will be more sensitive to
PPi concentration, perhaps allowing for a better control of
the level of this important metabolite. Knowledge of free
PPi concentration in cells possessing family II PPases is
required to address this point more specifically. B-PPase was initially
reported to be insensitive to fluoride (10), a well known inhibitor of
family I PPases, but more recent studies (13) have shown both families to be fluoride-sensitive.
A search through GenBankTM using B. subtilis
PPase as a template indicated 11 more verified and 3 unverified
full-length putative family II PPase sequences. The amino acid
sequences of Thermotoga maritima, Clostridium
difficile, and Geobacter sulfurreducens putative PPases
include a specific 230-residue-long insertion between residues Glu-67
and Val-68 (B-PPase numbering), indicating that their subunit size is
significantly larger than those of the other family II PPases (60 kDa
versus 33-34 kDa; Table V). However, the conservation
pattern of the amino acid residues strongly suggests that all 14 of
these sequences represent family II PPases. Interestingly, Vibrio
cholerae appears to be the first example of a species having genes
for PPases from both families. According to subunit size and dependence
on Mn2+, the PPase of Bacillus megaterium,
described by Tono and Kornberg (30), may also belong to family II. So
far, all family II PPase sequences belong to bacteria.
Quaternary Structure and Its Role in Catalysis--
Unlike the
bacterial PPases of family I, which are hexamers, the three PPases
studied in this work are dimers, dissociating into monomers at low
enzyme concentration in the absence of Mn2+. The dimeric
form, which is expected to be dominant under physiological conditions
(Mn2+ present), is much more active. The latter fact is at
variance with the data of Kuhn et al., who report B-PPase
(12) and M. jannaschii PPase (13) to exist as an inactive
dimer and active trimer. It should be noted that their molecular mass
estimates were obtained by gel filtration, a method more prone to error than the sedimentation analysis used in the present study. The dimeric
structure of Sm-PPase has recently been confirmed by x-ray crystallography (31)
The metal-free dimer is more active than the monomer due to increased
kcat and decreased Km values
(Table IV). With family I PPase from E. coli, dissociation
of the hexamer to trimers and dimers increases Km
without affecting kcat (27, 28). This result was
interpreted as evidence that hexamer formation and substrate binding
both induce a catalytically optimal structure for the active site. With
family II PPases, substrate binding is not sufficient to produce a
catalytically optimal structure, as indicated by decreased
kcat.
Role of Mn2+--
Family I PPases can utilize both
Mg2+ and Mn2+ as cofactors; however,
Mg2+ is more efficient in all cases. With family II PPases,
the efficiency of these cations as cofactors is reversed (Table IV),
and the available data indicate that this difference is due to a unique site, which binds Mn2+ with an affinity characteristic of
metalloenzymes. This site is quite specific for Mn2+
versus Mg2+; the ratio of the respective
KM1,obs values is 5,000-23,000 (Table III).
Mn2+ binding to this site controls activity in three ways.
First, it dramatically shifts the monomer
dimer equilibrium in the direction of the more active dimer (Kd changes more
than 105-fold; Table II). A similar but smaller effect is
exerted by Mg2+ (Kd changes
1,000-3,600-fold). Kuhn and Ward (12) also observed an effect of
Mn2+ and Co2+ on the quaternary structure of
B-PPase, consistent with metal ion binding to the high affinity site,
but interpreted this effect in terms of a dimer-trimer equilibrium.
Second, Mn2+ changes the kinetic parameters
kcat and Km for the dimer (Table IV). Interestingly, both kcat and
Km are increased in the presence of
Mn2+, but the effect on kcat is
larger. This means that switching from Mg2+ to
Mn2+ at the high affinity site would always activate the
enzymes, but the effect would be larger at higher substrate
concentration. The high-affinity site of the dimer obtained in the
absence of metal ions apparently binds Mg2+ from the assay
medium, which explains the similarity of the kinetic parameters for
this dimer and the dimer obtained in the presence of Mg2+
(Table IV). Finally, Mn2+ bound to the high affinity site
allows substantial activity with Ca2+ (Fig. 8). This means
that CaPPi is a reasonably good substrate for family II
PPases. By contrast, CaPPi is a strong, nonhydrolyzable inhibitor of family I PPases (29, 32, 33). At the low affinity sites
and in the PPi complex (true substrate), Mn2+
and Mg2+ appear to be equally effective in catalysis, as
indicated by similar activities of Mn2+-pretreated Sg-PPase
(i.e. containing Mn2+ at the high affinity
site) with Mn2+ and Mg2+ as activators (curves
Mn/Mn and Mn/Mg in Fig. 8). Kuhn and Ward (12)
report that Co2+ can activate B-PPase 70% as much as
Mn2+ by binding to the high affinity site, but no
activation was observed with other transition raw and alkali earth
metal ions.
Recent x-ray crystallographic studies (31) indicate a two-domain
structure for each subunit of dimeric Sm-PPase, the C-terminal domain
being quite flexible. The active site was located at the domain
interface and contained two protein-bound metal ions. One had three Asp
and one His, and the other had two Asp and one His side chains as
ligands. The four-ligand site apparently corresponds to the high
affinity site and the three-ligand site to one of the low affinity
sites detected in the present study. The other low affinity site(s)
appear(s) to have low occupancy in the crystals. In terms of the
three-dimensional structure, the effects of Mn2+ and
Mg2+ on dimer stability may result from decreased domain
flexibility caused by metal ion binding at the domain interface.
Cells of B. subtilis (34) and S. mutans (35)
accumulate large amounts of Mn2+. Hence this cation appears
to be a physiological ligand of family II PPases, at least at the high
affinity site, and the effects of Mn2+ reported above may
well be involved in PPase activity regulation in vivo. S. mutans has been identified as the primary cause of dental caries
(14) and manganese as a caries-promoting element (36) due to its
stimulation of S. mutans growth (37). Keeping in mind that
Mn2+ decreases the suppressive effect of fluoride both on
the activity of family II PPase (13) and on the growth of S. mutans (37), it is tempting to speculate that the action of
Mn2+ on S. mutans is due to its effect on
Sm-PPase. In in vitro studies of family II PPases, one
should keep in mind that the high affinity site may also bind other
metal ions (13) with unpredictable consequences for quaternary
structure and activity. Therefore parameters and factors such as the
concentration and nature of the added metal ion, enzyme concentration,
and purity of reagents should be carefully controlled in the solutions
used to store and assay these enzymes.