From the Merkert Chemistry Center, Boston College,
Chestnut Hill, Massachusetts 02467 and the § Department of
Biochemistry, Vanderbilt University School of Medicine,
Nashville Tennessee 37232
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ABSTRACT |
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The kinetics of PI-PLC Mammalian phosphoinositide-specific phospholipase C
(PI-PLC)1 enzymes are key
components of PI-mediated signaling cascades in vivo (1, 2).
These enzymes are subdivided into three main classes, PI-PLC- PI-PLC Given the structural complexity of this protein, kinetic studies with
well defined substrates can shed light on the roles and importance of
the different domains for the actual catalytic cleavage of PI. Although
purified PI-PLC Chemicals--
DiC7PC, POPC, POPA, LPA, POPS, PMPMe,
and PI were obtained from Avanti and used without further purification.
Triton X-100, SDS, oleic acid, GPI, GPIP, GPIP2, PIP, and
crude soybean PI were purchased from Sigma. Crude soybean PI (50% PI)
was used for the enzymatic generation (using bacterial PI-PLC) of cIP
as described previously (17).
Enzymes--
A rat cDNA encoding PI-PLC Preparation of Assay Solution--
The buffers used were 50 mM sodium acetate/acetic acid (pH 5.0), 50 mM
sodium citric/citric acid (pH 6.0), or 50 mM HEPES (pH 7.0, 7.5, or 8.0). A stock solution of cIP (200 mM) was prepared by dissolving cIP in D2O and adjusting the pH to 6.0. In
the cIP assays, 8 mM diC7PC, 8 mM
TX-100, and 24% organic solvent (Me2SO, dimethylformamide,
and isopropanol) were added to examine the interfacial activation and
organic solvent effect at different pH values. Mixed micelles of PI
solubilized in TX-100 or diC7PC were also used as
substrates for PI-PLC 31P NMR Assay of PI-PLC Water-soluble cIP Is a Substrate of PI-PLC
The dependence of enzyme activity on cIP concentration at pH 5.0 (Fig.
1B) is hyperbolic with a Km of 15 mM and a Vmax of 2.7 µmol
min Effect of Interfaces and Solvent on cIP Kinetics--
A kinetic
analysis of cIP hydrolysis by PI-PLC in the absence and presence of
interfacially active additives allows one to determine whether the
interfaces interact directly with the enzyme. In particular, if
interfaces enhance cIP hydrolysis, that interfacial activation must be
due to an allosteric transition of the enzyme rather than a change in
the substrate properties because cIP is water-soluble and has no
measurable tendency to associate with interfaces (17). This approach
was used to show that bacterial PI-PLC is allosterically activated by a
variety of PC aggregates (micelles and bilayers) (17) and that a
similar PC activation of PI-PLC
PA has been shown to enhance PI-PLC
Water-miscible organic solvents, such as 24% Me2SO,
dimethylformamide, or isopropanol, likewise inhibited PI-PLC PI Cleavage in TX-100 Mixed Micelles--
With naturally occurring
PI, detergents such as TX-100 are often used to form mixed micelles
that are good substrates for PI-PLC. Because TX-100 does not affect cIP
hydrolysis by PI-PLC
Inhibition of PI-PLC cleavage of PI by Ca2+ occurred
above a threshold at each pH, 1 mM at pH 5 and 0.5 mM at pH 7.5, when the ratio of TX-100 to PI was 2. This
inhibition by Ca2+ decreased when TX-100/PI = 6 (at
fixed PI) where considerably more detergent was present (Fig.
2B). The PI hydrolysis rate and light scattering by
TX-100/PI particles at 500 nm as a function of added TX-100 are shown
in Fig. 3 (A and
C). The maximum PI-PLC activity toward PI was reached at
TX-100/PI = 4, although the PI was well solubilized by a 2-fold
excess of TX-100 compared with PI (e.g. the reduced light
scattering at TX-100/PI = ~2). The requirement for increased
TX-100 suggests that PI molecules need to be well separated in TX-100
mixed micelles to obtain the highest activity. The need for separation
of PI molecules is balanced by "surface dilution" of the substrate
that gives rise to an apparent inhibition (25, 26). In contrast to
PI-PLC
The ratio of TX-100/PI was held constant and the concentration of PI
was varied to obtain an apparent Km for PI
solubilized in a TX-100 matrix. Although the apparent
Km extracted from this approach is a combination of
both a surface adsorption binding step and the true
KS value, it is a useful parameter for comparing the
effects of different interfaces on enzyme activity. For PI-PLC
Insight into the inhibition by high concentrations of Ca2+
was obtained by examining PI aggregation behavior as monitored by the
31P line width of PI in TX-100 micelles. If
Ca2+ induces aggregation of PI in TX-100 micelles (not
particle growth but clustering of PI), the line width of PI should
increase because the mobility of the clustered PI has been reduced. The
31P line widths for PI solubilized in TX-100 at different
ratios of PI to TX-100 (and mole fractions of PI) and Ca2+
concentration are shown in Fig. 4C. The PI line width
increased at Ca2+ concentrations greater than 1 mM for a TX-100/PI ratio of 2; however, the 31P
line width did not change much with TX-100/PI = 6. The increased line width for the higher mole fraction PI indicated a lower mobility for PI consistent with a Ca2+-induced aggregation. The
trend of increased aggregation of PI with increasing Ca2+
paralleled the decreased PI hydrolysis rate. For TX-100/PI of >4, the
lack of effect of Ca2+ on line width indicated that the PI
molecules are well separated and not as susceptible to
clustering/aggregation by Ca2+. Thus, a more dilute PI
surface may be a better substrate for PI-PLC PI Cleavage in diC7PC Mixed Micelles--
An excess of
diC7PC micelles inhibited PI-PLC The Effect of POPA and Other Interfacial
Additives--
Previously, it has been reported that PI-PLC Factors That Control cIP/I-1-P Partitioning--
Throughout the
time course of an individual assay, the ratio of PI-PLC
Assay temperature can also affect E·cIP stability (16, 22). The
specific activity of PI-PLC
Assay pH is another parameter that might also be expected to affect
cIP/I-1-P because the cIP hydrolysis step releases a proton. The ratio
of cIP/I-1-P decreased rapidly with increasing pH (Fig. 6B).
The inclusion of PA in the surface also affected the product ratio.
POPA not only increased the PI hydrolysis rate at pH values above 6.0 but also increased the ratio of cIP/I-1-P. PA in these micelles is a
monoanion below pH 5.5 and a dianion above pH 7. Perhaps it acts as an
interfacial buffer in facilitating proton release from the
protein upon cIP hydrolysis.
Mammalian PI-PLC isozymes are multidomain enzymes with key roles
in signal transduction by virtue of their generation of second messengers by substrate cleavage. A recurring theme is that their action can be modulated by interfaces interacting with several different domains (PH, C2, SH2, and SH3). Distinguishing kinetic enhancements caused by changes in surface properties from those caused
by allosteric effects on kcat is often difficult
with a phospholipid substrate. However, hydrolysis of cIP, the
water-soluble intermediate in the cleavage of PI, can be used as a
probe of allosteric effects of interfaces on PI-PLC enzymes. The high
Km of several PI-PLC enzymes for cIP compared with
PI suggests that an interfacial substrate binds more effectively to the
enzyme. Coupled with water-miscible organic solvent activation in the case of bacterial PI-PLC and PI-PLC PI cleavage by PI-PLC The pH dependence for PI-PLC1 toward a water-soluble
substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol
(PI) in detergent mixed micelles were monitored by
31P NMR spectroscopy. That cIP is also a substrate
(Km = ~15 mM) implies a two-step
mechanism (intramolecular phosphotransferase reaction to form cIP
followed by cyclic phosphodiesterase activity to form
inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLC
1 to form cIP
and I-1-P with the enzyme specific activity and ratio of products
(cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and
other amphiphilic additives. Cleavage of both cIP and PI by the enzyme
is optimal at pH 5. The effect of Ca2+ on PI-PLC
1
activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+
activates the enzyme; however, high Ca2+ inhibits
PI-PLC
1 hydrolysis of phosphoinositides (but not cIP) with the
extent of inhibition dependent on pH, substrate identity (cIP or PI),
substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLC
1 by high
Ca2+ is proposed to derive from the divalent metal
ion-inducing clustering of the PI and reducing its accessibility to the
enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid,
and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but
not at pH 5.0; they have no effect on cIP hydrolysis at either pH
value. These different kinetic patterns are used to propose a model for
regulation of the enzyme. A key hypothesis is that there is a
pH-dependent conformational change in the enzyme that
controls accessibility of the active site to both water-soluble cIP and
interfacially organized PI. The low activity enzyme at pH 7.5 can be
activated by PA (or phosphorylation by tyrosine kinase). However, this
activation requires lipophilic substrate (PI) present because cIP
hydrolysis is not enhanced in the presence of PA.
INTRODUCTION
Top
Abstract
Introduction
References
, -
,
and -
, that share three conserved regions (3): (i) an N-terminal
pleckstrin homology (PH) domain (these domains often have a high
affinity for the phosphoinositide PIP2), (ii) the X
domain (~170 amino acids), and (iii) the Y domain (~260 amino
acids). A C2 or Ca2+-lipid binding domain is also present
at the C-terminal end of PI-PLC-
and -
. In all cases, the X and Y
domains are necessary for catalysis (4) with the other domains involved
in regulating activity. For example, in the case of PI-PLC-
, the PH
domain plays an allosteric role in binding the protein to a bilayer
(5). PH domains also bind
G proteins very tightly (6), and
PI-PLC-
activity is regulated by interactions with Gq
proteins (7, 8).
isozymes are the largest of PI-PLC isozymes and are abundant
in many tissues and cell types. The biological significance of the
PI-PLC
1 enzyme has been documented (2). When PI-PLC
1 was
"knocked out" of mice by targeted gene disruption (9), the embryos
failed to develop beyond 9 days. The lethality was not due to a
specific organ failure but to a general lack of growth in all parts of
the embryo. Thus, in an intact mammal PI-PLC
1 is indispensable for
cell proliferation, and the requirement is not compensated for by other
signal transduction pathways or other PI-PLC isozymes. The enzymes are
activated by tyrosine kinase growth factor receptors (10, 11). The
phosphorylation enhances translocation of protein from cytosol to
plasma membrane (10). A unique feature of PI-PLC
isozymes that
distinguishes them from the
and
classes is that the extended
sequence between the X and Y domains is homologous to Src domains.
These SH2 and SH3 domains can interact with protein tyrosine kinases
and activate the enzyme (10-13). Flanking these Src homology domains
is a split PH domain as well. Deletion of SH2 and SH3 domains produces
an enzyme that still has catalytic activity but altered regulation (14). Expression of the X and Y domains in tandem leads to a folded
structure that has a 20-fold higher activity toward PI and now has
optimum activity at pH 7 (15).
1 exhibits a moderate preference for PIP2
as substrate (3), it can hydrolyze PI efficiently yielding both cIP and
I-1-P as products. In the present work we have focused on
characterizing the activity of PI-PLC
1 toward the water-soluble
substrate cIP as well as on PI/detergent mixed micelles. As was
observed for PI-PLC
1 (16), cIP is a substrate for PI-PLC
1, and
examining how nonsubstrate amphiphiles affect kinetic parameters is
useful in assessing how interfacial modulation of the protein affects
this catalytic step. Although PI-PLC
1 kinetics show some similarity
with PI-PLC
1, there are critical differences. In particular, the
observation of higher specific activity at pH 5 toward both
water-soluble cIP and PI, inhibition of PI cleavage by Ca2+
concentrations in excess of a critical threshold, the lack of surface
or organic solvent activation of the enzyme toward cIP, PA activation
toward PI (but not cIP) at pH >6, and changes in the ratio of product
cIP to I-1-P by temperature, pH, Ca2+, or other additives
provide insights into how this enzyme could be regulated in
vivo.
MATERIALS AND METHODS
1 was expressed as
a histidine-tagged fusion protein that was purified by Ni2+
agarose affinity chromatography to homogeneity (18).
1. The ratio of PI to TX-100 or
diC7PC was varied as indicated. The addition of PA, oleic
acid, SDS, and PMPMe to TX-100/PI mixed micelles was also examined. The
optimum Ca2+ concentration for cIP or PI hydrolysis appears
to be much higher than with phosphorylated water-soluble and
amphiphilic substrates; hence it is not necessary to buffer
Ca2+ in the assay solution. EGTA is not a good chelator for
Ca2+ at pH 5.0 and does not act as a buffer for free
Ca2+ in our assay solutions. Instead, the free
Ca2+ concentration was measured using the probe Fluo-3
(19). The background of Ca2+ in the assay solution was
around 10-20 µM. Additional Ca2+, ranging
from 0.1 to 6 mM, was added to the assay solution. The total volume of each assay was fixed to 350 µl.
1
Activity--
31P spectra were acquired at 202.7 Hz on a
Varian Unity 500 spectrometer using a 5-mm broadband probe.
31P NMR parameters were based on those used previously
(17). 31P chemical shifts were referenced to phosphoric
acid (5%) as an external standard. For all kinetic runs, a control
spectrum (t = 0 min) was acquired prior to the addition
of enzyme. The amount of enzyme added to initiate hydrolysis depended
on the substrate, pH, and other assay conditions. The reaction rate was
calculated from intensity changes of PI, cIP, or I-1-P. The
experimental error with this assay technique is ±10%.
RESULTS
1--
Bacterial
PI-PLC catalyzes the two discrete steps of PI cleavage sequentially
with accumulation of cIP occurring before conversion to I-1-P. This is
the result of facile product release and a high Km
(~90 mM without interfaces, as low as 25 mM
with interfaces present) and low Vmax (20 µmol
min
1 mg
1) for cIP, the substrate for the
cyclic phosphodiesterase step (17). In contrast, mammalian PI-PLCs
generate both cyclic and acyclic inositol phosphate products
simultaneously. If cIP is a substrate, the catalytic mechanism for the
mammalian enzyme must be sequential. As shown in Table
I, cIP is indeed a substrate for
PI-PLC
1, and the pH profile for the cyclic phosphodiesterase reaction is consistent with that reported for PIP2 cleavage
(15), with the highest enzyme activity observed at pH 5.0. Although Ca2+ was absolutely required for cIP hydrolysis, the
dependence of activity on Ca2+ was quite different from
that reported for other substrates such as PIP2 and PIP.
The maximum activity obtained toward cIP occurred with 2 mM
Ca2+; higher Ca2+ led to a slight decrease in
activity (Fig. 1A). In
contrast, with PIP2 or PIP as the substrate, the
Ca2+ requirement was much lower (100 µM), and
the PI-PLC specific activity reached a maximum and then decreased with
further increases in Ca2+ (20).
Activity of PI-PLC1 toward cIP (8 mM) at pH 5 (unless
otherwise noted) in the absence and presence of additives
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Fig. 1.
A, effect of Ca2+
concentration on PI-PLC 1 activity toward 8 mM cIP in 50 mM acetate, pH 5.0, and 30 °C. B, specific
activity of PI-PLC
1 as a function of cIP concentration in 50 mM acetate, pH 5.0, in the presence of 2 mM
Ca2+; the curve is the fit to the
Michaelis-Menten equation with Km = 15 mM and Vmax = 2.7 µmol
min
1 mg
1.
1 mg
1 obtained by fitting the data with
the Michaelis-Menten equation. The activity of the PI-PLC
1
preparation decreased slowly even when stored at
20 °C so that
this value for Vmax could be higher. The
Km of PI-PLC
1 for cIP was about half that for
full-length PI-PLC
1 (16). With a Km of 15 mM (and a lower Vmax than PI
cleavage), cIP levels would be expected to accumulate as PI is cleaved
and then decrease as I-1-P is generated as long as the release of cIP
into the solution is a facile event.
1 requires an intact PH domain (16).
In contrast to the large activation observed for bacterial PI-PLC or
PI-PLC
1, 8 mM diC7PC micelles inhibited
PI-PLC
1-catalyzed hydrolysis of cIP (8 mM) by 33%, and
8 mM TX-100 micelles had only a small activating effect
(15%) close to the error limit of the assays, both at pH 5 (Table I)
and at pH 7.5 (data not shown). Whatever the role of the PH, SH2, and
SH3 domains, any interactions they have with interfaces do not
appreciably affect the binding or hydrolysis of cIP.
1 activity toward
PIP2 (solubilized in TX-100 micelles) at pH 7.5 (21).
However, there was no enhancement of cIP hydrolysis in the presence of
short chain monomeric or micellar PA at either pH 5.0 or 7.5. Rather, inhibition of the cyclic phosphodiesterase activity was observed at
both pH values (Table I). The presence of low concentrations of
PIP2 (1 mM) more than sufficient to enhance
PI-PLC
1 hydrolysis of cIP (22) also failed to activate PI-PLC
1
toward 8 mM cIP.
1 at
both pH 5.0 and 7.5. Both dimethylformamide and isopropanol were
extremely potent inhibitors of the enzyme. This is in marked contrast
to the strong activating effect of these solvents on the cyclic
phosphodiesterase activity of bacterial PI-PLC (23) and PI-PLC
1
(16). For the latter PI-PLCs, the reduced solvent polarity is thought
to activate the enzyme by stabilizing a more active conformation of the
enzyme. The mixed solvents could mimic the polarity generated locally by interfaces such as PC bound to the enzyme. That neither PC nor
solvent activated PI-PLC
1 suggests that other factors are more
critical for activity of this enzyme.
1, it is unlikely to have an allosteric effect
on the enzyme. However, interactions of the detergent with PI and
cofactor Ca2+ can have pronounced effects on PI-PLC
kinetics by virtue of modifying the properties or accessibility of PI
in the interface. The reaction rates for PI-PLC
1 catalyzed
hydrolysis of 8 mM PI were measured as a function of
Ca2+ concentration at different ratios of TX-100 to PI at
both pH 5.0 and pH 7.5 (Fig.
2A shows this dependence for
TX-100/PI = 2). Similar to the pH dependence of cIP hydrolysis,
the rate of PI cleavage was about 20-fold higher at pH 5 than at pH
7.5. The optimal Ca2+ concentration also varied with pH. It
decreased when the pH was increased with a value of 0.5 mM
Ca2+ at pH 5.0 and 0.1 mM at pH 7.5. Product
distribution (cIP/I-1-P) also varied with pH. Both cIP and I-1-P were
observed at pH 5 with the ratio of products constant in a particular
assay. This behavior is not consistent with rapid release of cIP from
the enzyme because the Km for cIP is 15 mM. It suggests that PI-PLC
1, like PI-PLC
1, does not
readily release cIP generated in situ but holds it for
sufficient time so that the rate of attack by water is comparable with
its release (16, 24). Interestingly, there was no observable cIP
produced at pH 7.5; I-1-P was the only product detected from PI
cleavage.
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Fig. 2.
Ca2+ dependence of
PI-PLC 1 catalyzed hydrolysis of 8 mM PI solubilized in 16 mM TX-100 at pH 5.0 (
) and pH 7.5 (
) (A), 48 mM TX-100
at pH 5.0 (B), and 16 mM
diC7PC at pH 5.0 (C). The
specific activity was calculated from the decrease in intensity of the
PI intensity in the 31P NMR assay.
1, the bacterial PI-PLC acting on a fixed concentration of PI
solubilized in TX micelles exhibited a maximum activity at TX/PI = 1.5 (17) corresponding to conversion of the bulk of the
multilamellar vesicles to mixed micelles. As the amount of TX-100 was
increased, bacterial PI-PLC activity decreased, presumably because the
surface concentration of PI decreased.
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Fig. 3.
PI-PLC 1 catalyzed
cleavage of 8 mM PI as a function of added TX-100 in the
presence of 0.5 mM Ca2+ (A) or
diC7PC in the presence of 1.0 mM
Ca2+ at pH 5.0 (B). In C,
PI solubilization by TX-100 is monitored by light scattering at 500 nm.
1, the
apparent Km for PI decreased (from 4.3 to 2.8 mM) as the TX-100/PI ratio increased (Fig.
4, A and B),
suggesting that substrate molecules in the more dilute PI surface (0.14 versus 0.33 mole fraction PI) were more accessible to the
enzyme. The apparent Vmax also increased with
the more dilute PI surface (from 2.6 to 6.4 µmol min
1
mg
1), confirming that PI was a better substrate when more
dilute in TX micelles (e.g. at TX/PI = 6 or 0.14 mole
fraction versus TX-100/PI = 2 or 0.33 mole fraction).
The TX-100 enhanced activation must reflect a modification of substrate
in the interface to which the enzyme is sensitive, because TX-100 had
only minimal effects on cIP hydrolysis. Interestingly, cooperativity
was observed for PI cleavage (but not for cIP hydrolysis) by
PI-PLC
1. The lack of cooperativity for cIP hydrolysis strongly
suggests that cooperativity is not due to enzyme dimerization or
conformational change; rather it is related to substrate aggregation
and interface behavior.
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Fig. 4.
Specific activity at pH 5.0 of
PI-PLC 1 as a function of PI at fixed
TX-100/PI = 2 with 0.5 mM Ca2+
(A) and TX-100/PI = 6 with 1 mM
Ca2+ (B). The curves
represent fits to the Hill equation using a Hill coefficient of 2.5 and
with an apparent Km of 4.3 ± 0.4 mM and Vmax of 2.6 ± 0.2 µmol min
1 mg
1 at TX-100/PI = 2 and
an apparent Km of 2.8 ± 0.5 mM and
Vmax of 6.4 ± 0.4 µmol
min
1 mg
1 at TX-100/PI = 6. In
C is shown the line width of 8 mM PI in TX-100
micelles (at pH 5.0) as a function of Ca2+ concentration.
, TX-100/PI = 2;
, TX-100/PI = 6.
1 because it is less
susceptible to Ca2+-induced clustering.
1 activity toward cIP,
suggesting that some interaction of this lipid with the enzyme does
occur. Nonetheless, these short chain PC micelles can be used as a
matrix to solubilize PI for comparison to TX-100/PI mixed micelles. The
Ca2+ requirement for PI-PLC
1 cleavage of PI in
diC7PC mixed micelles is dramatically different from that
in TX mixed micelle as shown in Fig. 2C. PI solubilized in
the diC7PC matrix abolished the inhibition of PI-PLC
1 by
high concentrations of Ca2+ at both pH 5.0 and pH 7.5; the
cIP/I-1-P ratio was also significantly increased (Table
II). A likely explanation is that the
zwitterionic PC matrix prevents PI from Ca2+-induced
clustering, possibly by interacting with the Ca2+. The
activity of PI-PLC
1 toward PI solubilized in diC7PC
micelles was also much greater than toward PI in TX-100 micelles at pH 5.0. This apparent activation by PC is different from that occurring with bacterial PI-PLC and PI-PLC
because the PC interface has no
activating effect on cIP hydrolysis; rather there is a modest inhibition at this level of PC. The enzyme-catalyzed hydrolysis rate of
a fixed concentration of PI as a function of diC7PC was also different from that for PI solubilized in TX-100 micelles (Fig.
3B). The faster rate of PI hydrolysis in diC7PC
micelles and lower requirement for PC to solubilize the PI indicate
that in the assay mixture PI is better presented in a PC micelle than in a TX-100 mixed micelle. The kinetic effect is consistent with the
Ca2+-induced PI clustering, a physical effect associated
with reduced enzyme activity that is reduced in diC7PC
micelles.
Ratio of products (cIP/I-1-P) generated by PI-PLC1 catalyzed
cleavage of PI under different conditions
1 can be
activated by PA with the PA acting as an allosteric modifier in
reducing KS (21). The water-soluble phosphoester AMP
inhibited PI-PLC activity (21). AMP also inhibited PI-PLC
1 activity
toward cIP (Table I). However, neither PA solubilized in TX-100 or
diC7PC nor other interfaces were able to activate
PI-PLC
1 toward cIP hydrolysis. The effect of PA on PI hydrolysis
could be different, and effects of additives might shed light on the
complex interfacial behavior of this system. Therefore, several anionic
additives, POPA, lyso-PA, oleic acid, PMPMe, POPS, and SDS, were
examined for their effect on PI hydrolysis in TX-100 micelles. The
ratio of TX-100/PI/additives was 4.5:1:0.5, and the PI concentration was fixed at 8 mM; the assay pH was 7.5, with 0.1 mM Ca2+ added. The results are shown in Fig.
5. Additives POPA, lyso-PA, oleic acid,
and SDS enhanced PI hydrolysis to different extents, whereas PMPMe and
POPS had little effect. The PA-induced activation of PI-PLC
1
activity occurred in both TX-100/PI and diC7PC/PI mixed
micelles to about the same extent. Because PA activation was the
largest, it was examined as a function of Ca2+
concentration (from 0.02 to 0.5 mM) and pH; the surface
mole fraction of PA was 0.08, and the surface concentration of
substrate PI was 0.16. The maximum PI-PLC
1 activity was reached at
0.1 mM Ca2+; there was no significant change in
activity above 0.1 mM Ca2+ (data not shown).
Under these conditions, the presence of PA abolished the high
Ca2+ inhibition of PI hydrolysis. The pH profile of
PI-PLC
1 toward PI was also different in the presence and absence of
POPA at 0.3 mM Ca2+ (Fig.
6A). PA had little effect at
pH 5.0 and enhanced PI-PLC activity only above pH 6.0. The specific
activity of PI-PLC
1 toward PI in the presence of POPA at pH 7 was
within a factor of two of the specific activity at pH 5.0 in the
absence of PA. This represents a very large enhancement of PI-PLC
1
activity.
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Fig. 5.
Effect of anionic amphiphilic additives on
PI-PLC 1 catalyzed cleavage of PI in TX-100
micelles with 0.1 mM Ca2+, pH 7.5. The
ratio of TX-100/PI/additives was 4.5:1:0.5.
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Fig. 6.
The effect of pH on
PI-PLC 1 catalyzed cleavage of PI in TX-100/PI
micelles. A, specific activity. B, the ratio
of cIP to I-1-P, in the absence (
) and presence (
) of POPA. Assay
conditions include TX/PI/POPA = 5:1:0.5, with 8 mM PI
and 0.3 mM Ca2+.
1 products
(cIP/I-1-P) is constant, consistent with release of cIP and hydrolysis
of bound cIP to I-1-P occurring in parallel. However, cIP/I-1-P can be
altered by Ca2+ concentration, temperature, pH, or other
additives (e.g. PA). The cIP/I-1-P ratio decreased with
increasing Ca2+ concentration when PI was solubilized in
TX-100 at TX-100/PI = 2 (Fig.
7A). However, increased
Ca2+ had a much smaller effect on cIP/I-1-P when the mixed
micelles contained a more dilute surface concentration of PI
(e.g. TX-100/PI = 6). The ratio of cIP to I-1-P was
higher for TX-100/PI = 6 compared with TX-100/PI = 2; the
more dilute PI surface favored release of cIP into the solution
compared with attack by water to form I-1-P. The cIP/I-1-P product
ratio was also higher for PI solubilized in diC7PC micelles
(2:1 PC/PI). Because the ratio of cIP to I-1-P is likely to depend on
the residence time of the E·cIP complex produced in situ
(16, 22), any factors that alter the residence time of E·cIP will
affect the ratio of cIP to I-1-P. Interfaces can do this by altering
substrate or product exchange or partitioning at the surface as well as
by interacting directly with the protein.
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Fig. 7.
A, the ratio of cIP to I-1-P produced by
PI-PLC 1 cleavage of 8 mM PI in 16 mM TX-100
(pH 5.0) as a function of Ca2+. B, effect of
temperature on the rate of PI cleavage (
), generation of cIP (
),
and generation of I-1-P (
). C, the ratio of cIP to I-1-P.
PI (8 mM) was solubilized in 48 mM TX-100, pH
5.0, with 1 mM Ca2+.
1 was examined at several temperatures
(Fig. 7B). Because the apparent Km for PI in TX-100 micelles at TX-100/PI = 6 was 2.8 mM, the PI
concentration was fixed at 8 mM in the assay. At this
substrate concentration the activity was near its maximum value, and
specific activity approximated kcat. The
generation of I-1-P was less temperature-dependent than cIP
production from 10 to 30 °C, leading to an increase in cIP/I-1-P
with temperature (Fig. 7C); above 40 °C the enzyme began to lose activity. Transition state theory applied to data below 40 °C (ln (kcat/T)
versus 1/T is linear in that region) indicated that the
H
for release of cIP from
E·cIP compared with hydrolysis to I-1-P is +32 kJ/mol, whereas the
S
is +0.1 kJ/K-mol higher. These values are very
close to the
H
and
S
for PI-PLC
1 (16). Not surprisingly,
the entropy cost for generation of product I-1-P is high compared with
release of cIP, reflecting the energy cost for ordering a water
molecule to attack E·cIP.
DISCUSSION
1, the high Km
for cIP is likely to reflect difficulties in the enzyme binding a well
hydrated polar molecule. In striking contrast to those PLCs, PI-PLC
1
is more efficient in hydrolyzing cIP and does not exhibit solvent
activation. Because cIP structure is the same, the lower Km for cIP, lack of cooperativity in cIP binding,
and higher Vmax of PI-PLC
1 may reflect a
local decreased polarity at the active site. Another key kinetic
difference involves PH domains. The N-terminal PH domain of PI-PLC
1
was shown to interact allosterically with amphiphiles (PC or
PIP2), causing a 2-fold increase in
kcat toward cIP (16, 22). PI-PLC
1 has both an N-terminal domain and a split PH domain between the X and Y domains. Although there is significant sequence homology between the PH domains
of
1 and
1 isozymes, functional similarity is less obvious because there was no effect of PC or other interfaces (including PIP2 solubilized in TX-100 or diC7PC micelles)
on the cyclic phosphodiesterase reaction of PI-PLC
1. The PH domain
of PI-PLC
1 could still aid in anchoring the enzyme to an interface;
however, such interactions do not affect the catalytic machinery.
1 generates both cIP and I-1-P in parallel.
Given the kinetic parameters for cIP hydrolysis (which predict a
build-up of cIP prior to I-1-P generation), PI-PLC
1 is similar to
the
1 isozyme where the E·cIP complex has a relatively long lifetime. Because PI-PLC
1 also requires Ca2+ for
activity, the cIP may have a binding orientation at the active site
similar to that for a nonhydrolyzable cIP analog (27) binding to
PI-PLC
1 (24), where the phosphonate is a bidentate ligand of the
active site Ca2+. This arrangement would stabilize E·cIP.
However, Ca2+ has another more complex effect on the
phosphotransferase activity of PI-PLC
1 toward PI. Above a threshold
concentration, Ca2+ inhibits the first step of the
reaction; the cyclic phosphodiesterase step is not inhibited by high
Ca2+. The lack of effect on water-soluble cIP is consistent
with the Ca2+ affecting properties of the lipid substrate
(e.g. Ca2+-induced PI clustering) and not the
enzyme. Alternatively, if one invokes a secondary Ca2+ site
on the enzyme, then its occupation affects the phosphotransferase step
but not the cIP hydrolysis step. However, because additional nonsubstrate interfacial molecules (that do not affect cIP hydrolysis) reduce the high Ca2+ inhibition, the most reasonable
explanation is that high Ca2+ alters
substrate-Ca2+ interactions, and these changes inhibit
PI-PLC
1.
1 action on PI and cIP (roughly a
20-fold higher activity at pH 5 than 7) is also unusual and has
mechanistic implications. One or more key residues must be protonated
for optimal activity of both phosphotransferase and cyclic
phosphodiesterase activities of the full-length enzyme. Interestingly,
expression of X and Y domains without the SH2 or SH3 inserted domains
yields a highly active enzyme whose pH optimum is shifted to 7 (15). A
possible model for the pH dependence of PI-PLC
1 (Fig.
8) has the active site of PI-PLC
1
covered or occluded by the SH2-SH2-SH3 domains at pH 7; this "lid"
or cover would be displaced at pH 5 by protonation of key residues
(15). At pH 5, this SH2-SH2-SH3 lid is "open" and PI-PLC
1
activity is high; at pH 7, the lid is "closed" and the enzyme has
much lower activity. Consistent with this model is the previous
observation that protease V8 cleavage activates the enzyme at pH 7, and
the cleavage occurs within the lid between the SH3 and Y domains (28). Active enzyme at pH 5 can be inhibited by excess Ca2+
sequestering the substrate PI; this kinetic effect depends on the
detergent matrix used to solubilize PI. Ca2+ modulation of
the TX-100-PI interface also occurs at pH 7.
View larger version (26K):
[in a new window]
Fig. 8.
Proposed model for how pH, interfaces, and PA
affect PI-PLC 1. There is a conformational
change in the enzyme between pH 5 and 7.5 that controls accessibility
of the active site (the lid is open at pH 5 and closed at pH 7) to both
water-soluble cIP and interfacial PI. The "inactive" enzyme at pH 7 can be activated by PA or by receptor tyrosine kinase (TK)
phosphorylation (indicated by pY). The active enzyme at pH 5 can be inhibited by excess Ca2+ sequestering the substrate
PI; this latter effect depends on the detergent matrix used to
solubilize PI as well as on the Ca2+ concentration. This
Ca2+ modulation of the TX-100-PI interface also occurs at
pH 7.
The addition of anionic lipids (PA, LPA, SDS, and oleic acid) also
affects the pH profile, enhancing enzyme activity at pH 7.5. The
mechanism for the altered hydrolysis of PI could be due to the
amphiphiles interacting with either the enzyme or the substrate. One
possible explanation is that anionic lipids bind to the enzyme and open
the SH2-SH2-SH3 lid. Their presence should enhance both PI and cIP
cleavage at pH 7.5. Because only activity toward PI is enhanced,
substrate PI must also be needed to facilitate lid opening at pH 7. Alternatively, activation by PA and similar lipids could be the result
of altered substrate properties. The anionic amphiphiles could compete
with PI and lessen Ca2+-induced aggregation of substrate.
However, that is unlikely to account for (i) the very large activation
observed at pH 7.5 and (ii) the observation of the same magnitude of PA
activation with PI solubilized in diC7PC and TX-100
micelles (the TX-100 surface is much more susceptible to
Ca2+-induced clustering than the PC/PI surface, hence one
would expect a difference in the behavior of these two interfaces with
PA added). The pKa2 of micellar PA or LPA (either
short chain PA or long chain PA in TX-100 micelles) is ~7 (29). The
significant activation of PI-PLC1 around this value in the presence
of PA suggests that the PA dianion is critical for this effect.
PI-PLC
1 is regulated in vivo by phosphorylation (30, 31).
Phosphorylation might be another switch that alters the interaction of
the SH domains with the rest of the protein leading to activation of PI-PLC
1 at pH 7. Precedence for such interactions can be seen with
the SHP-2 structure (32). This offers the possibility of proteins
modulating PI-PLC
1 activity by interacting with the SH domains and
opening the (hypothetical) lid.
In summary, a model consistent with unusual kinetics of PI-PLC1
toward both water-soluble cIP and interfacially organized PI has been
proposed. It includes specific enzyme conformational changes due to pH
or nonsubstrate ligands as well as Ca2+-induced modulation
of interfacial substrate properties as ways to alter enzyme specific
activity. The model shown in Fig. 8 also makes several predictions that
can be tested in the future: (i) there should be a large conformational
change (presumably involving the SH domains) in the enzyme between pH 5 and 7 and (ii) because phosphonate esters have pKa
values shifted from phosphate esters, a phosphono-PA analog would be
expected to activate the enzyme but alter the pH profile of PI
hydrolysis in the presence of PA.
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ACKNOWLEDGEMENT |
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We thank Xiaoqing Qian for the initial work on cIP kinetics.
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FOOTNOTES |
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* This work has been supported by National Institutes of Health Grants GM 26762 (to M. F. R.) and CA75195 (to G. C.).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.
¶ To whom correspondence should be addressed. Tel.: 617-552-3616; Fax: 617-552-2705; E-mail: mary.roberts{at}bc.edu.
The abbreviations used are: PI-PLC, phosphatidylinositol-specific phospholipase C; PA, phosphatidic acid; PC, phosphatidylcholine; diCnPC, diacylphosphatidylcholine with n carbons in each acyl chain; POPA, 1-palmitoyl-2-oleoylphosphatidic acid; PI, phosphatidylinositol; PIP2, phosphatidylinositol-4,5-bisphosphate; PMPMe, 1-palmitoyl-2-myristoylphosphatidylmethanol; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; cIP, D-myo-inositol 1,2-(cyclic)-phosphate; I-1-P, D-myo-inositol-1-phosphate; TX-100, Triton X-100; PH, pleckstrin homology domain.
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REFERENCES |
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