Catalytic Domain of Phosphoinositide-specific Phospholipase C (PLC)
MUTATIONAL ANALYSIS OF RESIDUES WITHIN THE ACTIVE SITE AND HYDROPHOBIC RIDGE OF PLCdelta 1*

Moira V. EllisDagger , Stephen R. James§, Olga Perisicparallel , C. Peter Downes§, Roger L. Williamsparallel , and Matilda KatanDagger **

From the Dagger  Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, the parallel  Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, and the § Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Structural studies of phospholipase C delta 1 (PLCdelta 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 PLCdelta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (PLCbeta 1-beta 4, PLCgamma 1-gamma 2, and PLCdelta 1-delta 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 PLCbeta by G protein subunits and PLCgamma isozymes by tyrosine kinase-linked receptors are best understood.

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 PLCbeta , PLCgamma , and PLCdelta 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).

Critical to further understanding of PI-PLCs catalytic functions has been the determination of PLCdelta 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 PLCbeta , PLCgamma , and PLCdelta isozymes have all four domains found in PLCdelta 1 (17). Although PLCdelta 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 alpha /beta 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.

In this study, the data obtained from structural studies of PLCdelta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 PLCdelta 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 right-arrow 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 PLCdelta 1.

Mutations were introduced into PLCdelta 1 Arg60 right-arrow Lys by the two-stage PCR-based overlap extension method (20). The control PLCdelta 1 Arg60 right-arrow 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 right-arrow Ala, prepared by M13-based site-directed mutagenesis has been described previously (21). Mutants Arg549 right-arrow Ala, Ser522 right-arrow Ala, Trp555 right-arrow Ala, and Tyr551 right-arrow Ala were made using wild type primers 1 and 3; all others, excluding Asp343 right-arrow Arg and Glu390 right-arrow Lys, were made using wild type primers 1 and 2. Oligonucleotides used in these procedures are summarized in Table I.

                              
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Table I
Oligonucleotides used to generate PLCdelta 1 mutants
Primers with mutated nucleotides (underlined) and the wild type primers were used to introduce mutations into PLCdelta 1 by the two-stage PCR-based overlap extension method. Only forward primer sequences are shown for each mutant oligonucleotide.

Mutant fragments Asp343 right-arrow Ala, Glu341 right-arrow Ala, Glu341 right-arrow His, Glu341 right-arrow Gln, Glu390 right-arrow Ala, Glu390 right-arrow His, Glu390 right-arrow Gln, Phe360 right-arrow Ala, His356 right-arrow Ala, Arg438 right-arrow Ala, Lys440 right-arrow Ala, Leu320 right-arrow Ala, and Asn312 right-arrow Ala were digested with AccI/BsmI and subcloned individually into AccI/BsmI sites of PLCdelta 1 Arg60 right-arrow Lys. Mutant fragments Arg549 right-arrow Ala, Ser522 right-arrow Ala, Trp555 right-arrow Ala, and Tyr551 right-arrow Ala were digested with AccI/KpnI and subcloned individually into the AccI/KpnI sites of PLCdelta 1 Arg60 right-arrow Lys. All mutations were verified by sequencing, and each fragment was completely sequenced to confirm that no unwanted mutations had been created.

Expression and Purification of Recombinant Proteins-- Expression and purification of recombinant PLCdelta 1 protein lacking the first 57 amino acids residues has been described previously (19, 21).The same procedures were used to obtain PLCdelta 1 Arg60 right-arrow Lys and mutant enzymes. Briefly, glutathione S-transferase fusion protein was isolated from the Escherichia coli extract by affinity chromatography on glutathione-Sepharose. The PLCdelta 1 protein was separated from glutathione S-transferase and removed from the affinity matrix by thrombin cleavage. Soluble PLCdelta 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 PLCdelta 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 PLCdelta 1 enzymes were performed as described previously for beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Residues Selected for Site-directed Mutagenesis-- Based on the structure of PLCdelta 1 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 alpha /beta -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 PLCdelta 1 are invariant in all PI-PLCs. Other active site residues in PLCdelta 1 (Glu390, Lys438, Lys440, and Tyr551) are replaced conservatively in only a few sequences. Thus, Glu390 is replaced by aspartic acid only in PLCdelta 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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Fig. 1.   Schematic representation of the catalytic domain of phosphoinositide-specific phospholipase C. A ribbon representation of the PLCdelta 1 catalytic domain/IP3 complex as reported by Essen et al. (15). A, left panel, view looking down into the active site of the domain with the bound IP3 substrate analogue shown in ball-and-stick representation and the calcium co-factor for the reaction is shown as a large sphere; right panel, schematic representation for the interactions between the protein and the substrate analogue. Beneath each residue interacting with the substrate analogue are listed the mutants that were constructed and the specific activities of the mutants toward PIP2 (µmol/min/mg). B, left panel, view of the catalytic domain roughly 90° from the view shown in A. The loops forming the hydrophobic ridge at the rim of the active site opening are indicated. Right panel, expanded view of the hydrophobic ridge with hydrophobic side chains shown in ball-and-stick representation. The residues that were mutated are indicated.


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Fig. 2.   An alignment of the catalytic domain amino acid sequence from different PI-PLC isozymes. The catalytic domain sequences incorporating residues from the X and Y regions of sequence similarity have been compared; the linker region (X/Y linker) was omitted. The following sequences, described in Ref. 17, were compared: human PLCdelta 1 (delta 1 hum; residues 299-442 and 489-606), rat PLCdelta 1 (delta 1 rat; residues 299-442 and 489-606), bovine PLCdelta 2 (delta 2 bov; residues 293-437 and 487-603), rat PLCdelta 4 (delta 4 rat; residues 293-437 and 500-616), Drosophila melanogaster norpA (beta -like) PLC (beta  D.m; residues 322-471 and 546-633), rat PLCbeta 4 (beta 4 rat; residues 316-465 and 562-679), human retinal PLCbeta 4 (beta 4 hum; residues 163-312 and 408-525), D. melanogaster (beta -like) plc-21 (beta  D.m; residues 321-468 and 596-712), human PLCbeta 2 (beta 2 hum; residues 315-465 and 539-655), human PLCbeta 3 (beta 3 hum; residues 320-470 and 587-703), rat PLCbeta 1 (beta 1 rat; residues 319-469 and 537-653), bovine PLCbeta 1 (beta 1 bov; residues 319-469 and 537-653), soybean (Glycine max) delta -like PLC (delta  G.m; residues 109-252 and 330-447), A. thaliana delta -like PLC 2 (delta  A.t; residues 106-249 and 313-430), A. thaliana delta -like PLC 1 (delta  A.t; residues 108-250 and 290-407), D. discoideum delta -like PLC (delta  D.d; residues 325-467 and 536-650), rat PLCgamma 2 (gamma 2 rat; residues 315-458 and 927-1041), human PLCgamma 2 (gamma 2 hum; residues 315-458 and 927-1041), bovine PLCgamma 1 (gamma 1 bov; residues 323-466 and 950-1067), rat PLCgamma 1 (gamma 1 rat; residues 323-466 and 950-1067), human PLCgamma 1 (gamma 1 hum; residues 323-466 and 950-1067), Saccharomyces cerevisiae delta -like PLC (delta  S.c; residues 383-522 and 584-707), and Schizosaccharomyces pombe delta -like PLC (delta  S.p; residues 444-586 and 635-750). All-against-all comparisons of the sequences were performed using the Darwin suite of programs running on the Computational Biochemistry Research Group Server at Eidgenossische Technische Hochschule, Zurich (cbrg.inf.ethz.ch/welcome.html). Residues in PLCdelta 1 selected for mutagenesis are shown in bold. Invariant residues in all sequences are indicated by an asterisk. Secondary structure elements and residue numbers of PLCdelta 1 are also shown.

Structural studies of PLCdelta 1 (15) also revealed a cluster of hydrophobic residues around the active site opening (Fig. 1B). This hydrophobic ridge, located at one end of the active site opening, consists of a residue from helix alpha 5 and residues from three loops connecting beta 1 with alpha 1, beta 2 with alpha 2, and beta 7 and alpha 6. The hydrophobic residues include Leu320 in the loop beta 1/alpha 1, Tyr358 and Phe360 in the beta 2/alpha 2 loop, Trp555 in the beta 7/alpha 6 loop, and Leu529 from helix alpha 5. Comparison of PI-PLC sequences (Fig. 2) have shown that positions equivalent to PLCdelta 1 Leu320 are occupied by hydrophobic residues in all PI-PLCs. There is at least one hydrophobic residue in the loop beta 2/alpha 2 at positions corresponding to PLCdelta 1 Tyr358 and Phe360; Phe360 is better conserved with a hydrophobic residue absent in only two (bovine gamma 1 and human gamma 2) sequences. Conservation of hydrophobic residues equivalent to Trp555 is limited to only a few sequences. However, in most other sequences the position corresponding to residue 557, also present in the beta 7/alpha 6 loop, is occupied by hydrophobic residues and could also contribute to a hydrophobic ridge as described for PLCdelta 1.

All PLCdelta 1 active site residues and Leu320, Phe360, and Trp555 within the hydrophobic ridge were individually replaced, first by alanine and, in the case of Glu341, Glu390, and Asp343, by other residues as well (Table II). All changes have been introduced into the full-length PLCdelta 1 using PCR-based site-directed mutagenesis. The control enzyme and PLCdelta 1 with the point mutations were expressed as fusion proteins, subjected to thrombin cleavage, and further purified and concentrated on a Mono Q column. Expression levels of different point mutants were comparable to the control (~10 mg/liter of bacterial culture) with the exception of Ser522 that was reduced 5-10-fold.

                              
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Table II
Phospholipase C activity of control and mutant PLCdelta 1
Control PLCdelta 1 and different mutants were expressed in bacteria and purified to homogeneity. For calculation of specific activity, the activity of the control and mutant PLCdelta 1 were analysed using PIP2 as a substrate in the presence of 50 µM calcium (control conditions). Specific activities of control protein towards PIP2, PIP, and PI were determined at 50 µM calcium. The specific activity towards PIP2 was also determined in the presence of 3 mM EGTA. The data are averages of two measurements that did not differ more than 20%.

Effects of Replacements of the Active Site Residues on Activity of PLCdelta 1-- 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.

Data obtained from analysis of the active site residue replacements by alanine are summarized in Table II and Fig. 3. Specific activity of the control enzyme at a calcium concentration of 50 µM (control conditions) was about 1,000 units/mg, consistent with our previous measurements (19). The Glu341 right-arrow Ala mutation caused the greatest reduction of specific activity to 0.004 unit/mg, i.e. over 200,000-fold. The His311 right-arrow Ala, Asn312 right-arrow Ala, and Ser522 right-arrow Ala mutations resulted in reduction of specific activity to 0.03-0.09 unit/mg (10,000-35,000-fold). A reduction of 600-6,000-fold (specific activity 0.1-2 units/mg) was observed for the mutants Asp343 right-arrow Ala, His356 right-arrow Ala, Glu390 right-arrow Ala, Arg549 right-arrow Ala, and Tyr551 right-arrow Ala. Replacements of Lys438 and Lys440 resulted in only 30-fold and 5-fold reductions, respectively.


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Fig. 3.   Effect of replacement of active site residues by alanine on substrate hydrolysis. Purified proteins were analyzed for PIP2 (light hatched bars) and PI (black bars) hydrolysis under optimal conditions for the substrates. The data are averages of two measurements that did not differ more than 20% and are expressed as -fold reduction of control PLCdelta 1 activity.

The impact of the point mutations on activity of PLCdelta 1 was also analyzed using PI and conditions adjusted for maximum hydrolysis of this substrate (1 mM calcium, sodium deoxycholate mixed micelles) (Fig. 3). The -fold reductions of PI hydrolysis calculated for replacements of His311, Asn312, Glu341, Asp343, His356, Glu390, and Tyr551 to Ala were comparable with the data obtained for hydrolysis of PIP2 (all differences were less than 3-fold). In contrast, the specific activities of Lys438 right-arrow Ala, Lys440 right-arrow Ala, Ser522 right-arrow Ala, and Arg549 right-arrow Ala toward PI were not reduced to the levels measured with PIP2 as a substrate with the ratio PIP2/PI between 5- and 60-fold. Substrate specificity of these mutants toward PIP2, PIP, and PI were studied further (see below).

In addition to alanine replacements, Glu341 was also mutated into glutamine or histidine, Asp343 into arginine, and Glu390 into glutamine, histidine, or lysine (Table II). The impact of the histidine and glutamine replacements on PLC activity was similar to the effects of alanine mutations except for the Glu390 right-arrow Gln mutant, which had a slightly higher residual activity. In the case of the Asp343 right-arrow Arg and Glu390 right-arrow Lys mutations, where negative charges have been replaced by positively charged residues, activity was reduced an additional 30- or 80-fold, respectively.

Since a relatively large number of mutations have been made and many replacements greatly reduced the enzyme activity, a detailed kinetic analysis was performed only with the control PLCdelta 1, Glu390 right-arrow Ala, Arg549 right-arrow Ala, His356 right-arrow Ala, and His311 right-arrow Ala. Based on structural studies (15), these selected resides are likely to perform different functions. The main differences for all tested mutants were in the Vmax values. The Km values determined for the Glu390 right-arrow Ala (83 ± 15 µM), His356 right-arrow Ala (110 ± 21 µM), and His311 right-arrow Ala (74 ± 14 µM) were similar to the control (83 ± 17 µM), whereas the value determined for Arg549 right-arrow Ala was somewhat higher (125 ± 21 µM).

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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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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Fig. 4.   Mutations that selectively affect hydrolysis of PIP2 and PIP compared with hydrolysis of PI. Activity of control and mutant PLCdelta 1 was determined using PIP2 (light hatched bars), PIP (dark hatched bars), or PI (black bars) as a substrate at 50 µM calcium. The data are expressed as -fold reduction compared with the control PLCdelta 1 (A). Ratio of -fold reduction of PIP2 and PI hydrolysis (PIP2/PI) and PIP and PI hydrolysis (PIP/PI) is also shown for each mutant (B).

Calcium Dependence of Substrate Hydrolysis-- It has been shown previously that activity of PLCdelta 1 (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 PLCdelta 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 right-arrow Ala, Lys438 right-arrow Ala, Lys440 right-arrow Ala, Ser522 right-arrow Ala, Arg549 right-arrow Ala, and Tyr551 right-arrow 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 PLCdelta 1 calcium dependence (Fig. 5). Some differences have been also observed for the His311 right-arrow Ala mutant, which shows less inhibition of activity at high calcium concentrations than the control enzyme. The activity of the Asp343 right-arrow Ala and Asn312 right-arrow Ala mutants at 1 mM calcium was comparable to their activities at concentrations of 50 µM, whereas for Glu341 right-arrow Ala and Glu390 right-arrow Ala the peak of the activity was shifted to 1 mM calcium. A similar shift was observed for the Asp343 right-arrow Arg and Glu390 right-arrow 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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Fig. 5.   PLCdelta 1 mutations showing changes in Ca2+ requirement for PIP2 hydrolysis. Preparations of purified proteins were diluted to similar PLC activity as determined under control conditions and then analyzed at different calcium concentration for PIP2 hydrolysis. Activity of each mutant is expressed as percent of hydrolysis at 50 µM calcium.

Analysis of the control PLCdelta 1 has shown that the enzyme prepared and analyzed in the presence of 3 mM EGTA had a specific activity reduced to about 0.06 unit/mg (Table II). Under these conditions, it is still possible that some calcium remained bound to PLCdelta 1 and that the complete removal of the metal could have an even more pronounced effect. Based on these observations, it is expected that mutation of residues essential for the calcium binding would result in a great reduction of the enzyme activity. All mutations of Asn312, Glu341, Asp343, and Glu390 residues (in particular, Glu341 right-arrow Ala, Asp343 right-arrow Arg, and Glu390 right-arrow Lys) had a large impact on the activity (1,000-200,000-fold) (Table II). It is however difficult to assess the relative contribution of each of these residues, since a role of some residues may not be limited to calcium binding (see "Discussion").

Mutations within the Hydrophobic Ridge-- Analyses of the Leu320 right-arrow Ala, Phe360 right-arrow Ala, and Trp555 right-arrow 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).

For further studies of these mutants, the possibility that the hydrophobic ridge could penetrate into a phospholipid membrane was examined by measuring PIP2 hydrolysis in monolayers at different surface pressures. Fig. 6A shows that increasing monolayer surface pressure in PC/PS/PIP2 monolayers (70:27:3 by molarity) was accompanied by a decline in PIP2 hydrolysis catalyzed by the control PLCdelta 1. The decrease in rate of hydrolysis was linear with about a 5-fold difference between activity at the surface pressure of 10 mN/m and 35 mN/m. Analysis of the mutants in this system, in contrast to the mixed micelle assay, revealed an increase in activity compared with the control. Furthermore, as illustrated for the Phe360 right-arrow Ala mutant (Fig. 6B), activity was less dependent on increasing surface pressure. The surface pressure/activity relationship was also analyzed by measurements of the induction time tau  between addition of the enzyme to the subphase and onset of catalysis. The induction time measured for the control PLCdelta 1 was greater at higher monolayer pressures (Fig. 6C). It has been suggested that in this assay system the penetration rate (kp) would be inversely proportional to the tau  time (13). Therefore, the data for the control PLCdelta 1 would suggest that increasing surface pressure reduces the penetration rate into the monolayer. When PLCdelta 1 enzymes with the point mutations within the hydrophobic ridge were compared with the control, it was found that the tau time of Trp555 right-arrow Ala and Leu320 right-arrow Ala mutants was less dependent on monolayer pressure and that the Phe360 right-arrow Ala mutant was independent of the surface pressure (Fig. 6C).


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Fig. 6.   Effects of replacements of the hydrophobic ridge residues on PLCdelta 1 activity in a monolayer assay. A, pressure-activity relationship of the control PLCdelta 1 in mixed phospholipid monolayers. PC/PS/PIP2 monolayers containing 33P-labeled PIP2 were spread as described previously (13, 26) and 200 ng of enzyme added after 5 min. Reaction times were 15 min, and the rate of PIP2 hydrolysis was determined from linear portions of continuous trace recordings of radioactivity in the monolayer. Data points are the mean ± range of duplicate monolayers, except 10 and 35 mN/m, which were taken from single experiments. B, the Phe360 right-arrow Ala mutant was analyzed as described in A. C, induction times for PIP2 hydrolysis in PC/PS/PIP2 mixed monolayers by the control and three hydrophobic ridge point mutants of PLCdelta 1. Induction times (tau ) were derived by computer-aided integration of trace recordings of 33P-labeled PIP2 hydrolysis by the different enzymes at all surface pressures examined, as described previously (13). Each data point is the mean (± range or S.D.) of at least two determinations of tau  from each assay performed. Circles, control PLCdelta 1; inverted triangles, Trp555 right-arrow Ala; squares, Leu320 right-arrow Ala; triangles, Phe360 right-arrow Ala.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we describe the first systematic analysis of amino acid replacements within the PLCdelta 1 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>PIP>> PI has been observed. However, some differences between PI-PLC families have been found, such as lower ratio of PIP2/PI hydrolysis for PLCgamma than for PLCbeta and PLCdelta (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 PLCdelta 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 PLCdelta 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 right-arrow 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 PLCbeta 2 (residues 457-464) identified Lys461 and Lys463, corresponding to Lys438 and Lys440 in PLCdelta 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 PLCbeta 2 was analyzed only with PIP2 as a substrate. Replacements of four arginine residues within the Y region of conserved sequences in human PLCdelta 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 right-arrow Ala mutant, also somewhat reduced PI hydrolysis, whereas Arg549 right-arrow 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 PLCdelta 1 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 PLCdelta 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 PLCdelta 1) had been analyzed. The replacement of Glu341 in PLCdelta 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 PLCdelta 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 alpha /beta 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 PLCdelta 1 mutant that is calcium-independent like the bacterial enzyme, mutations Glu390 right-arrow Lys and Asp343 right-arrow 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 PLCdelta 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 PLCdelta 1. 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 PLCdelta 1 Leu356 mutant (35) and a replacement of the corresponding His380 in PLCgamma 1 to phenylalanine (41). However, three candidate residues in PLCdelta 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 PLCdelta 1 His311, and the corresponding His335 in PLCgamma 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 right-arrow Gln mutant would be able to coordinate the metal but would not function as a nucleophile. The mutational analysis of PLCdelta 1 (Table II) has shown about a 100-fold greater impact of the Glu341 right-arrow Ala replacement compared with the Glu390 right-arrow 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 right-arrow Gln mutation resulted in significant residual activity compared with the Gln390 right-arrow 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 PLCdelta 1), 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 PLCbeta , PLCgamma , and PLCdelta activity as the pressure increases. The surface pressure/activity relationship observed for PLCdelta was less complex than for PLCbeta and PLCgamma , where changes of the pressure within a small range resulted in a dramatic reduction of the activity (11). A more linear relationship for PLCdelta 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 PLCdelta 1) inserts into monolayers in a work-requiring step prior to activation (12). The surface area of the PLCdelta 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.

    ACKNOWLEDGEMENT

We thank Damian Counsell for help with the sequence alignments.

    FOOTNOTES

* 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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