From the Structural studies of phospholipase C
Hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PIP2)1 to the
second messengers inositol 1,4,5-trisphosphate and diacylglycerol by
phosphoinositide-specific phospholipase C (PI-PLC) is one of the
earliest key events in the regulation of various cell functions by a
number of extracellular signaling molecules (1-3). Three families of
mammalian PI-PLCs with 10 different isozymes (PLC Studies of the catalytic properties of PI-PLC revealed that, unlike
regulatory mechanisms, all eukaryotic enzymes have some common
characteristics (4). The catalytic activity is strictly dependent on
calcium as a cofactor and increases with a rise of calcium
concentrations within the physiological range (0.01-10 µM). Phosphatidylinositol (PI), phosphatidylinositol
4-monophosphate (PIP), and PIP2 are hydrolyzed with a
preference for PIP2 and PIP, but the enzymes are unable to
hydrolyze 3-phosphoinositides. There is a high stereospecificity for
the D-myo-inositol configuration of the
headgroup but not for the configuration of the C-2 position of the
diacylglycerol moiety (5). Although glycerophosphorylinositol phosphates can be hydrolyzed by PI-PLC (6), the presence of at least
short lipid side chains is required for the efficient catalysis (7).
Kinetic studies of some PLC Critical to further understanding of PI-PLCs catalytic functions has
been the determination of PLC In this study, the data obtained from structural studies of PLC Materials
Oligonucleotides for mutagenesis and sequencing were supplied by
Oswel DNA Services and also made in-house using an Applied Biosystems
DNA synthesizer (model 394). PCR reagents and the ABI PRISM dye
terminator cycle sequencing ready reaction kit for automated sequencing, using an ABI 377 sequencer, were from Perkin-Elmer. Wizard
PCR preps were from Promega and mini-prep kits from Qiagen. PGEX-2T
vector, glutathione-Sepharose 4B, and Mono Q column (PC1.6/5) were from
Amersham Pharmacia Biotech. Human thrombin, sodium salts of soybean PI,
bovine brain PIP and PIP2, as well as dioleyl
phosphatidylcholine (PC) and Folch extract of bovine brain (for
purification of PIP2 for the monolayer assay) were
purchased from Sigma. Pig brain phosphatidylserine (PS) was from Doosan
Serdary Research Laboratories. Phosphatidyl([3H])inositol
([3H]PI) and Phosphatidyl [3H]inositol
4,5-bisphosphate ([3H]PIP2) were obtained
from NEN Life Science Products. Phosphatidylinositol 4-phosphate
(inositol-2-3H) ([3H]PIP) was obtained from
American Radiolabeled Chemicals Inc. [33P]ATP (used for
synthesis of [33P]PIP2 with partially
purified PIP kinase from rat brain) was from Amersham Pharmacia
Biotech.
Methods
Plasmid Construction and Site-directed Mutagenesis--
The
2.2-kilobase pair BamHI/SmaI fragment from rat
brain PLC Cancer Research Campaign Centre for Cell and
Molecular Biology,
Medical Research Council Laboratory of Molecular
Biology,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 (PLC
1) in complexes with the inositol-lipid headgroup and
calcium identified residues within the catalytic domain that could be
involved in substrate recognition, calcium binding, and catalysis. In
addition, the structure of the PLC
1 catalytic domain revealed a
cluster of hydrophobic residues at the rim of the active site opening
(hydrophobic ridge). To assess a role of each of these residues, we
have expressed, purified, and characterized enzymes with the point
mutations of putative active site residues (His311,
Asn312, Glu341, Asp343,
His356, Glu390, Lys438,
Lys440, Ser522, Arg549, and
Tyr551) and residues from the hydrophobic ridge
(Leu320, Phe360, and Trp555). The
replacements of most active site residues by alanine resulted in a
great reduction (1,000-200,000-fold) of PLC activity analyzed in an
inositol lipid/sodium cholate mixed micelle assay. Measurements of the
enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bis-phosphate
(PIP2) identified Ser522, Lys438,
and Arg549 as important for preferential hydrolysis of
polyphosphoinositides, whereas replacement of Lys440
selectively affected only hydrolysis of PIP2. When PLC
activity was analyzed at different calcium concentrations,
substitutions of Asn312, Glu390,
Glu341, and Asp343 resulted in a shift toward
higher calcium concentrations required for PIP2 hydrolysis,
suggesting that all these residues contribute toward Ca2+
binding. Mutational analysis also confirmed the importance of His311 (~20,000-fold reduction) and His356
(~6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2
hydrolysis in the mixed-micelles, resulted in an enzyme that was less
dependent on the surface pressure when analyzed in a monolayer. This
systematic mutational analysis provides further insights into the
structural basis for the substrate specificity, requirement for
Ca2+ ion, catalysis, and surface pressure/activity
dependence, with general implications for eukaryotic
phosphoinositide-specific PLCs.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1-
4, PLC
1-
2, and PLC
1-
4) have been characterized; PI-PLC
molecules from other eukaryotes have properties shared with mammalian
isozymes from one of the main families. Regulation of PI-PLCs has been extensively studied and reveals several distinct mechanisms that link
multiple isozymes to various receptors (1). Among those mechanisms, the
activation of mammalian PLC
by G protein subunits and PLC
isozymes by tyrosine kinase-linked receptors are best understood.
, PLC
, and PLC
isozymes have also
demonstrated that the membrane associated isozymes can catalyze several
cycles of PIP2 hydrolysis functioning in a processive mode
of catalysis (8-10), and, when analyzed in monolayer assays, show
activity dependence on the monolayer surface pressure (11-14).
1 crystal structure (15, 16). This
revealed a four-domain organization of the enzyme consisting of a
pleckstrin homology (PH) domain, an EF-hand domain, a catalytic domain,
and a C2 domain. An alignment of PI-PLC sequences has suggested that
PLC
, PLC
, and PLC
isozymes have all four domains found in
PLC
1 (17). Although PLC
1 shares sequence similarity with other
PI-PLC throughout its sequence, the regions with the highest sequence
similarity are contained within the catalytic domain. The residues
within the conserved region X and the most conserved part of the Y
region (4) form two halves of the catalytic
/
barrel (15). Based
on structural studies of complexes with the PIP2 headgroup
and the catalytic calcium, the importance of individual residues within
the catalytic domain for the substrate binding, catalysis, and membrane
interactions has been suggested (15). Furthermore, the crystal
structure of the complex with cyclic inositol phosphate (18) and
kinetic studies (6) have suggested a reaction mechanism for PI-PLC catalysis. The data support general acid/base catalysis in a sequential mechanism with cyclic inositol phosphate as a reaction
intermediate.
1
complexes with the ligands have been used as a framework for a
structure/function analysis. The residues that constitute putative
active site and several hydrophobic residues in the vicinity of the
active site opening have been subjected to site-directed mutagenesis.
The impact of individual replacements on the PLC activity was analyzed
under different conditions to establish further structural requirements
for substrate recognition, calcium binding, catalytic steps, and
surface pressure dependence.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 cDNA (19) was subcloned into PGEX-2T vector, which
encodes the enzyme as a glutathione S-transferase fusion
protein. A BamHI site had been engineered at the 5' end of
the cDNA to enable cloning into the PGEX-2T vector; an internal
BamHI site had first been mutated by PCR without changing
the amino acid sequence. A mutation has also been introduced by PCR at
residue 60, an internal thrombin cleavage recognition site, from
arginine to lysine (Arg60
Lys) resulting in higher
yields of the glutathione S-transferase fusion protein and
its cleavage by thrombin only at the engineered cleavage site. This
mutation did not interfere with any other function of the enzyme, and
this mutant will be referred to as the control PLC
1.
1 Arg60
Lys by the
two-stage PCR-based overlap extension method (20). The control PLC
1 Arg60
Lys was used as the template for the first stage
PCR reaction; gene fragments with overlapping complementary ends from
the first round were paired to provide templates for the second round
using wild type external primers. One mutant, His311
Ala, prepared by M13-based site-directed mutagenesis has been described
previously (21). Mutants Arg549
Ala, Ser522
Ala, Trp555
Ala, and Tyr551
Ala
were made using wild type primers 1 and 3; all others, excluding
Asp343
Arg and Glu390
Lys, were made
using wild type primers 1 and 2. Oligonucleotides used in these
procedures are summarized in Table I.
Oligonucleotides used to generate PLC1 mutants
1 by the two-stage
PCR-based overlap extension method. Only forward primer sequences are
shown for each mutant oligonucleotide.
Expression and Purification of Recombinant
Proteins--
Expression and purification of recombinant PLC1
protein lacking the first 57 amino acids residues has been described
previously (19, 21).The same procedures were used to obtain PLC
1
Arg60
Lys and mutant enzymes. Briefly, glutathione
S-transferase fusion protein was isolated from the
Escherichia coli extract by affinity chromatography on
glutathione-Sepharose. The PLC
1 protein was separated from
glutathione S-transferase and removed from the affinity
matrix by thrombin cleavage. Soluble PLC
1 was further purified from
minor contaminants by chromatography on a Mono Q column (PC 1.6/5)
using a SMART system (Amersham Pharmacia Biotech). Determination of
protein concentration was according to Bradford (22), using bovine
serum albumin as a standard, and aliquots of purified protein (5-10
mg/ml) stored at
20 °C. Electrophoresis in SDS-acrylamide gels,
performed according to Laemmli (23), showed that purity of all PLC
1
proteins was >90%. In most cases, the protein yield was about 10 mg/liter of starting bacterial culture.
PLC Activity in Mixed Micelles-- The assay of hydrolysis of PIP2 and PI was based on methods described previously (19, 21, 24, 25). The standard reaction mixture for PIP2 hydrolysis contained 50 mM Tris-HCl, pH 6.8, 100 mM NaCl, 0.5% sodium cholate, 5 mM 2-mercaptoethanol, 0.4 mg/ml bovine serum albumin, 220 µM PIP2 (0.025 µCi), and CaHEDTA buffer for the final concentration of free calcium of 50 µM. Incubation was at 37 °C for 10 min. In this assay, 1 unit of PLC activity corresponds to hydrolysis of 1 µmol of PIP2/min. The same assay conditions were used to monitor hydrolysis of PIP and PI. In addition, PI hydrolysis was also analyzed in sodium deoxycholate mixed micelles (0.05% sodium deoxycholate) in the presence of 1 mM calcium. The calcium dependence of PIP2, PIP, and PI hydrolysis was analyzed using a range of calcium buffers as described previously (24).
For kinetic analysis of control and mutant enzymes, initial velocities were measured at PIP2 concentrations of 0.055, 0.110, 0.220, 0.440, and 0.660 mM, with incubation times of 0, 2.5, 5, 10, and 20 min. Apparent Km and Vmax values were determined by plotting results as the double reciprocal Lineweaver-Burk plot.PLC Activity in Monolayers--
Monolayer assay of the activity
of the control and mutant PLC1 enzymes were performed as described
previously for
-isoforms of PLC (13, 14, 26). The composition of the
monolayers was 70% PC, 27% PS, 3% PIP2 by molarity,
which were spread over a buffer comprising 10 mM Hepes, pH
7.2, 120 mM KCl, 10 mM NaCl, 2 mM
EGTA, 1 mM MgCl2, and 1 µM free
Ca2+ ions. Enzyme (200 ng) was added to the subphase, via
an injection port in the Teflon trough, 5 min after the monolayer was
spread to allow the surface pressure to stabilize. PIP2
hydrolysis was assayed for 15 min, during which time the monolayer
radioactivity was assayed continuously as described previously.
Induction times were determined by computer-aided integration of the
trace recording of changes in radioactivity in the monolayer, and the
extent of the reaction was determined by sampling the radioactivity
remaining in the monolayer and in 1 ml of subphase buffer at the end of the reaction.
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RESULTS |
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Residues Selected for Site-directed Mutagenesis--
Based on the
structure of PLC1 complexes with inositol phosphates and/or calcium
analogues (15, 18), the following residues have been implicated in
interactions with the ligands: His311, Asn312,
Glu341, Asp343, His356,
Glu390, Lys438, Lys440,
Ser522, Arg549, and Tyr551 (Fig.
1A). These putative active
site residues are present within a broad, solvent accessible depression
on the C-terminal end of the catalytic
/
-barrel. Comparison of 23 sequences of PI-PLC from mammalian sources and other organisms such as
slime mold, yeast, and plants demonstrated that these residues are well
conserved among eukaryotic enzymes (Fig.
2). Residues corresponding to
His311, Asn312, Glu341,
Asp343, His356, Ser522, and
Arg549 in PLC
1 are invariant in all PI-PLCs. Other
active site residues in PLC
1 (Glu390,
Lys438, Lys440, and Tyr551) are
replaced conservatively in only a few sequences. Thus,
Glu390 is replaced by aspartic acid only in PLC
4,
Lys438 by serine in plant PI-PLCs, Lys440 by
histidine in the enzyme from Dictyostelium discoideum, and Tyr551 by phenylalanine in Arabidopsis thaliana.
This strict conservation of residues that could comprise the PI-PLC
active site suggests that substrate recognition and mechanism of
catalysis are likely to be common to all eukaryotic enzymes.
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Effects of Replacements of the Active Site Residues on Activity of
PLC1--
PLC activity of the enzymes with single point mutations
of the active site residues was compared with the activity of the control enzyme using a sodium cholate/PIP2 mixed micelle
assay. In previous studies using this assay system (19, 21, 27), it was
demonstrated that the deletion of the PH domain (containing the high
affinity, non-catalytic PIP2-binding site) had no effect on
the rate of PIP2 hydrolysis and that the remainder of the
enzyme could directly bind and hydrolyze the substrate presented in
this way.
|
Mutations Affecting Substrate Specificity--
Among mutations
that differentially affected hydrolysis of PIP2 and PI,
Lys438, Arg549, and Ser522 have
been implicated in interactions with the 4-phosphoryl group and
Lys440 in interaction with the 5-phosphoryl group of the
inositol ring. To distinguish whether the reduction in PIP2
hydrolysis resulted from a loss of interactions with the 4- or
5-phosphate, we compared the PLC activity of these mutants toward PI,
PIP, and PIP2, prepared as sodium cholate mixed micelles,
in the presence of 50 µM calcium (Fig.
4). Activity of the Lys440
Ala mutant was reduced (~5-fold) only with PIP2 as a
substrate. The specific activity of this mutant was similar to the
specific activity of the control enzyme toward PIP (Table II),
consistent with Lys440 interaction with the 5-phosphoryl
group. The Lys438
Ala mutation resulted in a reduction
of both PIP2 and PIP hydrolysis (15-20-fold) with very
little effect on PI hydrolysis. The specific activity of this mutant
was comparable with the activity of the control enzyme using PI as a
substrate. The mutation Arg549
Ala had an effect on
hydrolysis not only of PIP2 and PIP but also PI. Both the
hydrolysis of PIP2 and PIP were greatly reduced relative to
hydrolysis of PI. This ratio (PIP2/PI and PIP/PI) for
Arg549
Ala was the most pronounced (about 50-fold)
among the analyzed mutants. The effect of this mutation on PI
hydrolysis could be due to an additional interaction with the
2-hydroxyl of the inositol (Fig. 1A) that would affect
hydrolysis of all inositol-lipid substrates. In the case of
Ser522
Ala replacement, the effect on PI hydrolysis was
a reduction of about 1,000-fold with a further decrease in specific
activity (5-10-fold) toward PIP and PIP2.
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Calcium Dependence of Substrate Hydrolysis--
It has been shown
previously that activity of PLC1 (as well as other PI-PLC enzymes)
has different calcium dependence curves when analyzed with
PIP2 and PI as a substrate presented as sodium cholate or
sodium deoxycholate micelles (19). The specific activity of the control
PLC
1 used in this study toward PIP2 was ~300 units/mg at 0.5 µM calcium, ~1,000 units/mg at 50 µM calcium, and ~140 units/mg at 1 mM
calcium. The specific activity toward PI in a similar assay system
increased within the tested range of calcium concentrations (0.5 µM to 10 mM) and was ~7 units/mg at 0.5 µM calcium, ~18 units/mg at 50 µM
calcium, and ~25 units/mg at 1 mM calcium. Although
simpler relationship of calcium dependence was observed with PI than
with PIP2 as a substrate, the reduced activity of the
mutants made the measurements difficult with this less efficiently
hydrolyzed substrate. Therefore, the activity of the control and mutant
enzymes with the point mutations of the active site residues was
compared at different calcium concentrations (0.5 µM to
10 mM) using PIP2 as a substrate. The control
(Fig. 5) and His356
Ala,
Lys438
Ala, Lys440
Ala,
Ser522
Ala, Arg549
Ala, and
Tyr551
Ala mutants (data not shown) had a similar
calcium dependence of PIP2 hydrolysis with the maximum at
about 50 µM calcium and decreasing at higher calcium
concentrations. Mutations of Asn312, Glu341,
Asp343, and Glu390, however, resulted in
changes of PLC
1 calcium dependence (Fig. 5). Some differences have
been also observed for the His311
Ala mutant, which
shows less inhibition of activity at high calcium concentrations than
the control enzyme. The activity of the Asp343
Ala and
Asn312
Ala mutants at 1 mM calcium was
comparable to their activities at concentrations of 50 µM, whereas for Glu341
Ala and
Glu390
Ala the peak of the activity was shifted to 1 mM calcium. A similar shift was observed for the
Asp343
Arg and Glu390
Lys mutants (data
not shown). Residues Asn312, Glu341,
Asp343, and Glu390 have been implicated in
calcium binding (Fig. 1A), and this difference in PLC
dependence of calcium concentrations further supports their proposed
role.
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Mutations within the Hydrophobic Ridge--
Analyses of the
Leu320 Ala, Phe360
Ala, and
Trp555
Ala mutants in PIP2/sodium cholate
mixed micelles at 50 µM calcium have shown that these
mutants, in comparison with the mutants in the active site, had little
effect; they resulted in a small reduction in PIP2
hydrolysis (Table II). Similar data have been obtained using the
substrate/dodecyl maltoside mixed micelles, and measurements of
Km values in this assay revealed that enzymes with mutations in the hydrophobic ridge behaved similarly to the control enzyme, having largely unaltered interfacial Km
values (data not shown).
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DISCUSSION |
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In this study, we describe the first systematic analysis of amino
acid replacements within the PLC1 catalytic domain based on the
crystal structure of this enzyme. The data provide further evidence for
a relationship between the individual residues and the catalytic
properties of PI-PLC enzymes (including substrate specificity,
requirement for Ca2+ ion, and surface pressure/activity
dependence) with implications for all eukaryotic PI-PLCs.
It is generally accepted that agonist stimulation of PI-PLC activity
can result in preferential hydrolysis of polyphosphoinositides and, in particular, PIP2 (see, e.g., Ref. 28).
Although presentation of PIP2 in cells could contribute
toward a preference for this inositol lipid, it has been shown that the
substrate selectivity is, to at least some extent, a property of PI-PLC
enzymes (4). Characterization of PI-PLC enzymes in vitro
revealed that all eukaryotic enzymes, unlike bacterial PI-PLC, can
hydrolyze PIP2 and PIP. Generally, a preference
PIP2>PIPPI has been observed. However, some
differences between PI-PLC families have been found, such as lower
ratio of PIP2/PI hydrolysis for PLC
than for PLC
and
PLC
(29). The degree of preference for polyphosphoinositides described for the same isozyme also varies considerably depending on
the conditions and substrate presentation used in different assays
in vitro (30-33). Some of these differences could be due to
composition and concentration of detergents and metal ions affecting
directly hydrolysis by the enzyme and also substrate presentation of
different inositol-lipids. Within a multidomain structure of eukaryotic
PI-PLCs, some of the domains could interact non-catalyticaly with the
inositol lipids and (depending on the assay conditions) also influence
the rate of substrate hydrolysis (8, 10). In this study, we used mixed
micelles of each inositol-lipid substrate with sodium cholate and found
that the PLC
1 specific activity with PIP2 as a substrate
was about 3-fold higher than with PIP and about 50-fold higher than
with PI (Table II). The ratio of substrate hydrolysis in this assay was
not affected by a deletion of the PH domain, eliminating a contribution
of this high affinity non-catalytic PIP2 binding site. The
structural studies of the PLC
catalytic domain (15, 18) implicated
Ser522, Lys438, and Arg549 in
interactions with the 4-phosphate and suggested their importance for
preference of both PIP and PIP2 over PI. The only residue that could interact with the 5-phosphate, Lys440, emerged
as a candidate residue that could provide specificity for
PIP2. Replacements of each of these residues by alanine
resulted in selective changes of PIP2, PIP, and PI
hydrolysis consistent with their predicted functions (Fig. 4). Among
these mutations, the greatest impact on preference for
polyphosphoinositides was the Arg549
Ala replacement.
However, replacements of some residues, most notably
Ser522, not only selectively reduced hydrolysis of
polyphosphoinositides, but also significantly reduced hydrolysis of PI
suggesting their role in some other functions of the enzyme or their
importance for the protein stability. Replacement of
Tyr551, implicated in hydrophobic interactions with the
sugar ring of inositol, equally reduced hydrolysis of PIP2
and PI (Table II and Fig. 3). Several of the residues implicated in
substrate binding and selectivity have been analyzed previously. A
study of the conserved region rich in basic residues in PLC
2
(residues 457-464) identified Lys461 and
Lys463, corresponding to Lys438 and
Lys440 in PLC
1, as important for PIP2
hydrolysis (34). It is, however, difficult to compare those data and
this study further, since a different assay system has been used and
PLC
2 was analyzed only with PIP2 as a substrate.
Replacements of four arginine residues within the Y region of conserved
sequences in human PLC
1 identified Arg549 as selectively
important for PIP2 compared with PI hydrolysis (30, 35).
The replacement of this residue with glycine, as with our
Arg549
Ala mutant, also somewhat reduced PI hydrolysis,
whereas Arg549
His mutation only affected
PIP2 hydrolysis (30).
Kinetic analysis of PI and PIP2 hydrolysis by PI-PLC enzymes, in the assay systems where preference for PIP2 was clearly observed, revealed only small differences in the apparent Km values (24, 31-33, 36). Consistent with these observations, the main effect of Arg549 replacements, which selectively reduced hydrolysis of polyphosphoinositides, was on Vmax (Ref. 30 and this report). It is therefore possible that additional interactions with the 4- and 5-phosphoryl groups of the inositol ring in PIP2 have little effect on the affinity for this substrate. However, these interactions could greatly reduce flexibility of inositol headgroup movements within the active site, resulting in higher hydrolytic efficiency. Recent structural studies using an inositol monophosphate support this possibility (18).
Structural studies and kinetic analysis of both eukaryotic and
bacterial enzymes have outlined a common mechanism of substrate hydrolysis: general acid/base catalysis with formation of cyclic inositol in a phosphotransfer step, followed by its conversion to an
acyclic inositol in a phosphohydrolysis step (5, 37). However, a
distinct characteristic of substrate hydrolysis by all eukaryotic
enzymes is dependence on Ca2+ as a cofactor. Structural
studies of PLC1 have suggested that the principal function of the
Ca2+ is to lower the pKa of the
2-hydroxyl group of the inositol moiety so as to facilitate its
deprotonation and subsequent nucleophilic attack on the 1-phosphate. A
second role of the metal could be to stabilize the negatively charged
transition state (18). Although all eukaryotic enzymes require
Ca2+, some differences in calcium dependence have been
observed among different PI-PLCs and among inositol-lipid substrates
(24, 29, 32, 33, 36). Generally, maximum hydrolysis of PIP2
was achieved at 5-50 µM calcium and further increase in
calcium concentrations (1-10 mM) had an inhibitory effect.
Crystallographic studies of complexes of PLC
1 with Ca2+
and PIP2 headgroup (IP3) revealed complex
interactions with the catalytic Ca2+ involving several
negatively charged residues in the active site (Glu390,
Glu341, and Asp343), Asn312, and
2-hydroxyl group of IP3 (15, 18). Replacement of these residues by alanine resulted in a great reduction of the enzyme activity (1,000-200,000-fold) (Table II). Furthermore, calcium dependence of these mutants was shifted toward higher calcium concentrations (Fig. 4) showing the importance of all these residues in
coordination of calcium. In previous studies, based on sequence alignments, only one of these residues (Glu341 in PLC
1)
had been analyzed. The replacement of Glu341 in PLC
1 by
glycine resulted in a loss of the enzyme activity; the calcium
dependence of this mutant, however, was not analyzed (35). The same
replacement has been found in the p130 protein, containing X and Y
regions found in PI-PLCs with the closest similarity to PLC
1, but
without detectable PI-PLC activity (38).
The acidic residues that coordinate the catalytic Ca2+ in
eukaryotic PI-PLCs are not present in the enzyme from Bacillus
cereus. Instead, basic residues (Arg69 and
Lys115) are present in the positions equivalent to
Asp343 and Glu390 within a similar /
barrel structure (37, 39). It has been suggested that these basic
residues, like Ca2+ in eukaryotic enzymes, facilitate
nucleophilic attack and stabilize the resulting transition state (37,
39). A recent mutational analysis of Arg69 is consistent
with this proposal (40). In our attempts to generate a PLC
1 mutant
that is calcium-independent like the bacterial enzyme, mutations
Glu390
Lys and Asp343
Arg (Table II) as
well as the double mutant (data not shown) have been made. However,
these mutations did not reduce calcium dependence, possibly due to
small structural differences between the active sites of eukaryotic and
the bacterial enzymes, which could still be too complex to allow this
conversion. Another reason for the inability to convert PLC
1 into a
calcium-independent enzyme by these mutations could be that the role of
calcium is not restricted to a positive charge. The structure of the
enzyme in complexes with intermediate analogues suggests that the
Ca2+ makes additional ligations with the transition state
that may sterically accelerate catalysis (18).
An effort has also been made to clarify the identity of residues
important for the phosphotransfer and phosphohydrolysis steps. It has
been suggested that in PI-PLC from B. cereus
His32 and His82 act as general base/acid
catalysts (37, 39). A residue in eukaryotic enzymes that could have an
equivalent role to His82 is likely to be the conserved
histidine corresponding to His356 in PLC1. A replacement
of this residue by alanine resulted in a great reduction of the enzyme
activity (Table I). Similar observations have been reported previously
for PLC
1 Leu356 mutant (35) and a replacement of the
corresponding His380 in PLC
1 to phenylalanine (41).
However, three candidate residues in PLC
1, His311,
Glu390, and Glu341, have been considered for
the role of His32 from the bacterial enzyme, which is
located within hydrogen bonding distance of the 2-OH group of the
inositol and could deprotonate the hydroxyl in a step leading to the
formation of the cyclic intermediate (15, 37). Mutational analysis in
this (Table II) and previous studies (21, 35, 41) identified PLC
1
His311, and the corresponding His335 in
PLC
1, as an important catalytic residue. In the structural studies,
using inositol phosphates that mimic the binding of substrates and the
reaction intermediates, His311 appears to be too distant
and unfavorably oriented for hydrogen-bonding with the 2-hydroxyl group
of any of the studied inositol phosphates (18). The structural data are
more consistent with the notion that His311 is essential
for the stabilization of a pentavalent transition state. Alternative
candidates for the general acid/base catalyst, Glu341 and
Glu390, are not only the calcium ligands but also form
hydrogen bonds to the 2-OH group of the inositol. A proton transfer
between either of the glutamate residues and the 2-hydroxyl group would
be feasible even in the presence of the positively charged calcium,
because any change in the partial charge of the carboxyl group would be compensated by an opposing charge at the 2-hydroxyl. An example for a
glutamate residue acting both as a metal ligand and as a nucleophile is
Glu70 in inositol monophosphatase. In addition to
structural data supporting these roles of Glu70 (42, 43),
it has been shown that the mutation of this residue to glutamine
decreased kcat dramatically while leaving metal
binding unaffected (44). This is consistent with the prediction that the Glu70
Gln mutant would be able to coordinate the
metal but would not function as a nucleophile. The mutational analysis
of PLC
1 (Table II) has shown about a 100-fold greater impact of the
Glu341
Ala replacement compared with the
Glu390
Ala mutation. To analyze the function of these
two glutamate residues further, replacements to glutamine were also
made; this mutation would eliminate the function of a general acid/base
catalyst. Since the Glu390
Gln mutation resulted in
significant residual activity compared with the Gln390
Ala mutant, the Glu341 residue would be more likely to act
as the general base of the first step of the reaction.
Studies of eukaryotic (8-10) and bacterial (45) PI-PLC enzymes
suggested that interactions with the membrane involve interactions additional to the substrate binding in the active site. Although some
of these sites in eukaryotic enzymes are clearly outside the catalytic
domain (e.g. the PH domain of PLC1), the hydrophobic ridge at the rim of the active site opening could provide a
non-catalytic membrane interaction site for this domain (17, 46, 47). Experiments where dependence of PLC activity on surface pressure was
examined in lipid monolayers have shown a decrease of PLC
, PLC
,
and PLC
activity as the pressure increases. The surface pressure/activity relationship observed for PLC
was less complex than for PLC
and PLC
, where changes of the pressure within a small range resulted in a dramatic reduction of the activity (11). A
more linear relationship for PLC
observed in this study (Fig. 6A) is in agreement with the the data reported previously
(11, 12). One possible interpretation offered for the surface pressure dependence was that a part of the PLC molecule (estimated to be ~1
nm2 in PLC
1) inserts into monolayers in a work-requiring
step prior to activation (12). The surface area of the PLC
1
hydrophobic ridge is roughly consistent with the area that could
penetrate into the membrane. The possibility that the residues present
in the hydrophobic ridge could underlie this activity/surface pressure relationship was supported by the observation that the replacements of
these residues by alanine-generated enzymes that were less dependent on
the surface pressure (Fig. 6, B and C). The
greatest change is caused by the replacement of Phe360, the
hydrophobic residue most exposed in this area. These data (Fig.
6C) have also shown that at high surface pressure (30-35 mN/m), when the packing of phospholipids is believed to be comparable to that in biological membranes, the enzymes with the reduced hydrophobic surface had shorter induction times than the control, suggesting a negative rather than positive contribution of the hydrophobic ridge to the rate of substrate hydrolysis. Thus, overcoming such a negative influence potentially could play a part in the activation of PI-PLC's in vivo.
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ACKNOWLEDGEMENT |
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We thank Damian Counsell for help with the sequence alignments.
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FOOTNOTES |
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* This work was supported by grants from the Cancer Research Campaign (to M. K.), the Medical Research Council/DTI/ZENECA LINK Program (to R. L. W.), and the British Heart Foundation (to R. L. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Pharmacia and Upjohn AB, 112 87 Stockholm, Sweden.
** To whom correspondence should be addressed. Tel.: 44-171-352-8133; Fax: 44-171-352-3299; E-mail: matilda{at}icr.ac.uk.
1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PLC, phospholipase C; PI-PLC, phosphoinositide-specific phospholipase C; PCR, polymerase chain reaction; PH, pleckstrin homology; PC, phosphatidylcholine; PS, phosphatidylserine; N, newton(s); IP3, inositol 1,4,5-trisphosphate.
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REFERENCES |
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