©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Structure-Function Study of Bovine Pancreatic Phospholipase A Using Polymerized Mixed Liposomes (*)

(Received for publication, September 28, 1994; and in revised form, November 2, 1994 )

Rajiv Dua Shih-Kwang Wu Wonhwa Cho (§)

From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A new combinatorial approach that includes the genetic variation of protein structure and the chemical modification of phospholipid structure in polymerized mixed liposomes was used to delineate the structure-function relationships in the interfacial catalysis of bovine pancreatic phospholipase A(2) (PLA(2)). Based on previous structural and mutational studies, several bovine PLA(2) mutants were generated in which a positive charge of putatively important lysyl side chains was reversed (K10E, K53E, K56E, and K116E) or neutralized (K56Q and K116Q). Kinetic parameters of bovine wild type and mutant PLA(2)s determined using polymerized mixed liposomes consisting of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (or -phosphoglycerol) and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol showed that Lys-53 is involved specifically in the interaction with a substrate bound in the active site. Also, these results showed that Lys-10 and Lys-116 are involved in the interaction of bovine PLA(2) with anionic interfaces but not in the interaction with the active site-bound substrate. In particular, Lys-116 makes more significant contribution than Lys-10 by 1.0 kcal/mol to the binding to anionic interfaces. Most importantly, Lys-56 was shown to participate in the interaction with both the active site-bound substrate and anionic interfaces. These findings establish Lys-56 and Lys-116 as essential residues for the binding of bovine pancreatic PLA(2) to anionic interfaces. Lastly, our structure-function analysis based on the use of polymerized mixed liposomes was further supported by equilibrium binding measurements of these proteins using 1,2-bis[12-(lipoyloxy)dodecanoyl]sn-glycero-3-phosphoglycerol polymerized liposomes and by kinetic analyses using monomeric substrates, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine and -phosphoglycerol.


INTRODUCTION

Phospholipase A(2) (PLA(2); (^1)EC 3.1.1.4) catalyzes, mostly in a Ca-dependent mode, the hydrolysis of the fatty acid ester in position 2 of 3-sn-phospholipids (for a recent review, see Dennis(1994)). In general, the PLA(2)-catalyzed reaction is described as a two-step process, the initial binding to the membrane surface (interfacial binding) and subsequent catalytic steps (Jain and Berg, 1989). Also, it has been generally believed that PLA(2) has an interfacial binding site separated from the active site (for a recent review, see Scott and Sigler(1994)). Recent structural analyses of PLA(2)-inhibitor complexes for some secretory PLA(2)s have revealed the interactions of a phospholipid molecule with active site residues of enzyme (Scott et al., 1990a, 1990b, 1991; Thunnison et al., 1990; White et al., 1990). However, the structural basis for the interfacial binding and the identity of protein residues involved in this process are relatively poorly understood due to difficulties inherent in determining the structure of membrane-bound proteins. Recent structure-function studies on bovine and porcine pancreatic PLA(2)s by site-directed mutagenesis have provided valuable information about the role of individual protein residues in the function of these enzymes (Dupureur et al., 1992a, 1992b; Kuipers, et al., 1989a, 1989b, 1990; Lugtigheid et al., 1993a, 1993b; Noel et al., 1990, 1991; Van den Berg et al., 1988). In many cases, however, an intimate interplay between the interfacial binding step and catalytic steps in the PLA(2) catalysis made it difficult to clearly delineate structure-function relationships from kinetic analyses using conventional PLA(2) substrates. Recently, we have developed a novel kinetic system using polymerized mixed liposomes that allows an unambiguous distinction between phospholipids interacting with the active site of PLA(2) and ones interacting with the interfacial binding site of PLA(2) (Wu and Cho, 1993, 1994). In this report, we present kinetic and binding studies of bovine pancreatic PLA(2) and its selected mutant proteins using polymerized mixed liposomes. These studies unambiguously identify protein residues that are involved in the interfacial binding and the substrate binding in the active site and evaluate their relative contributions to individual processes.


EXPERIMENTAL PROCEDURES

Materials

1,2-Dihexanoyl-sn-glycero-3-phosphoethanolamine (diC(6)PE) and -phosphoglycerol (diC(6)PG), 1,2-dioctanoyl-sn-glycero-3-phosphoglycerol (diC(8)PG) were purchased from Avanti Polar Lipids. 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (pyrene-PE), and -glycerol (pyrene-PG) were from Molecular Probes. 1,2-Bis[12-(lipoyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -glycerol (BLPG) were prepared as described previously (Wu and Cho, 1993). Large unilamellar liposomes were prepared by multiple extrusion through 0.1-µm polycarbonate filter (Millipore) in a microextruder Liposofast (Avestin, Ottawa, Ontario) (MacDonald et al., 1991). Phospholipid concentrations were determined by phosphate analysis (Kates et al., 1986). Fatty acid-free bovine serum albumin was from Miles Inc. 5,5`-Dithiobis(2-nitrobenzoic acid) and sodium sulfite were obtained from Aldrich. 2-Nitro-5-(sulfothio)-benzoate was synthesized from 5,5`-dithiobis(2-nitrobenzoic acid) according to the procedure of Thannhauser et al.(1984). Phenylmethanesulfonyl fluoride, TPCK-treated trypsin, and oxidized and reduced glutathione were purchased from Sigma. Urea was from Fisher. All of the restriction enzymes, T4 ligase, T4 polynucleotide kinase, and isopropyl-1-thio-beta-D-galactopyranoside were obtained from Boehringer Mannheim. An expression vector for bovine pancreatic PLA(2) (pTO-propla2) (Deng et al., 1990) was kindly provided by Dr. Ming-Daw Tsai. Oligonucleotides were purchased from Midland Co. (Midland, TX).

Construction of Mutant Phospholipase A(2) Genes

The site-directed mutagenesis of bovine pancreatic PLA(2) was performed using the M13mp18 vector containing bovine PLA(2) gene according to the method of Nakamaye and Eckstein(1986) using an in vitro mutagenesis kit from Amersham Corp. The oligonucleotides used for the construction of mutant proteins were 5`-ATCTTACATTCGATCATTC-3` (K10E), 5`-TTAGCTTGTTCATAGCAAT-3` (K53E), 5`-ATCAAGTTTTTCAGCTTGTT-3` (K56E), 5`-ATCAAGTTTCTGAGCTTGTT-3` (K56Q), 5`-TATCAAGATTTTCGTGTTCTT-3` (K116E), 5`-TCAAGATTCTGGTGTTCTT 3` (K116Q), respectively, in which underlined bases indicate the location of mutation. After DNA sequences of mutants were verified by the sequencing analysis (Sanger et al., 1977) using a Sequenase 2.0 kit (U. S. Biochemical Corp.), individual recombinant PLA(2) genes were digested with EcoRI and BamHI and inserted into pTO-propla2 vector.

Protein Expression and Purification

Wild-type and recombinant proteins were expressed and purified as described by Noel et al.(1991) with some modifications. An Escherichia coli strain BL21(DE3)pLysS (Novagen) was used as a host for the protein expression. Typically, cultures were grown in 3 times 1 liter of Luria broth containing chloramphenicol (0.02 mg/ml) and ampicillin (0.25 mg/ml). When the absorbance of media at 600 nm reached 0.8, additional ampicillin was added to a final concentration of 0.5 mg/ml, and the protein expression induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside to a final concentration of 0.5 mM. After 5 h, cells were harvested, and the bacterial pellet was suspended in 60 ml of 0.1 M Tris-HCl buffer, pH 8.0 containing 5 mM EDTA, 0.5% (v/v) Triton X-100. Sonication using a Sonifier 450 (Branson) was performed at 300 watts by a pulse mode for 6 times 1 min, and the solution was allowed to stir for 30 min at room temperature. The inclusion body pellet was obtained by centrifugation for 15 min at 10,000 times g at 4 °C. The pellet was solubilized in 6 ml of 8 M urea solution containing 0.3 M sodium sulfite, pH 8.0, and stirred vigorously at room temperature for 30 min. Two ml of 2-nitro-5-(sulfothio)-benzoate solution (25 mM) was then added, and the modification was monitored spectrophotometrically at 412 nm. After the modification was complete (10 min), the mixture was further stirred for 30 min and eventually centrifuged at 20,000 times g for 1 h at 4 °C. The supernatant was applied to a Sephadex G-25 (Pharmacia Biotech Inc.) column (2.5 times 20 cm) equilibrated in 25 mM Tris-HCl buffer, pH 8.0, containing 5 M urea and 5 mM EDTA, and the protein peak was collected and dialyzed against water and then against 0.3% (v/v) acetic acid to precipitate the sulfonated protein. The precipitated protein was washed with 120 ml of water and resuspended in 30 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM EDTA and 8 M urea. The solution was slowly diluted to 120 ml by the addition of 90 ml of 50 mM Tris-HCl, pH 8.0, containing 8 mM reduced glutathione, 4 mM oxidized glutathione, and 5 mM EDTA, kept at room temperature for 20 h, dialyzed against 50 mM ammonium bicarbonate, and eventually lyophilized. The protein was then dissolved in 5 ml of 10 mM Tris-HCl, pH 8.0, and centrifuged at 10,000 times g for 10 min at 4 °C to remove all insoluble proteins. TPCK-treated trypsin was then added to a final concentration of 0.2% (w/w), and the solution was stirred at room temperature. The PLA(2) activity, monitored using pyrene-PG/BLPG polymerized mixed liposomes as a substrate (Wu and Cho, 1994), typically reached a maximum after 2 h. Phenylmethanesulfonyl fluoride was then added to a final concentration of 0.1 mM, and the solution was dialyzed against 10 mM ammonium acetate, pH 5.0. The solution was filtered through 0.2-µm filters and applied to a Mono S column (Pharmacia) attached to a fast protein liquid chromatography system (Pharmacia). The column was equilibrated in 10 mM ammonium acetate, pH 5.0, and eluted with a linear gradient of increasing NaCl concentration from 0 to 0.5 M in the same buffer. Typically, PLA(2) was eluted as a single peak with 0.08-0.1 M of NaCl. The PLA(2) peak was collected, dialyzed against water, and lyophilized. The lyophilized protein was stored at -20 °C. Purity of protein was confirmed by SDS-polyacrylamide electrophoresis and isoelectric focusing using a Phast system (Pharmacia). Protein concentrations were determined by the Micro BCA method (Smith et al., 1985) (Pierce). Circular dichroism (CD) spectra of proteins were measured at room temperature using a Jasco J-600 spectropolarimeter. Each spectrum was obtained from 10 scans between 195 and 250 nm.

Kinetic Measurements

All of the kinetic experiments were performed at 37 °C and at pH 7.4. Kinetics of PLA(2)-catalyzed hydrolysis of polymerized mixed liposomes were performed according to the procedure previously described (Wu and Cho, 1993). Typically, the hydrolysis was performed in 1 ml of 10 mM HEPES buffer, pH 7.4, containing 0.5 µM pyrene-containing phospholipid inserted in 9.5 µM BLPG, 10 µM bovine serum albumin, 0.16 M NaCl, 0.1 mM EDTA, and 10 mM CaCl(2). Detailed descriptions of the experimental basis for selecting this kinetic condition were reported elsewhere (Wu and Cho, 1994). Phospholipid concentration in the range of 10-50 µM (in total concentration) did not affect the rate constants. The progress of hydrolysis was monitored as an increase in fluorescence emission at 380 nm (F) using a Hitachi F4500 fluorescence spectrometer with the excitation wavelength set at 345 nm. Spectral bandwidth was set at 5 nm for both excitation and emission. In order to obtain kinetically meaningful rate constants for the comparison of enzymatic activities while avoiding complicated kinetic analyses, we employed the simplest possible kinetic model that is consistent with the observed kinetic patterns. Under the above experimental conditions, the hydrolysis of pyrene-containing phospholipid/BLPG polymerized mixed liposomes by bovine PLA(2) and most of mutants followed the first-order kinetics (Fig. 1). Thus, we treated enzymatic reactions as a simple bimolecular process as shown in ,


Figure 1: The hydrolysis of pyrene-PG (0.5 µM)/BLPG (9.5 µM) polymerized mixed liposomes by bovine wild-type PLA(2) (4 nM) and K56E mutant (0.4 µM) in 10 mM HEPES buffer, pH 7.4, containing 10 mM bovine serum albumin, 0.16 M KCl, 0.1 mM EDTA and 10 mM CaCl(2). The reaction curves were reconstructed from data collected in a digitized form. The slope of lines indicates the initial velocity, and arrows indicate the initiation of reaction.



where E, S, P(1) and P(2), and (k/K(m)) represent free enzyme, phospholipid, fatty acid, lysophospholipid, and apparent specificity constant, respectively. Accordingly, the entire course of reaction was analyzed using a simple integrated first-order rate equation; i.e. [P] = S(o)bullet(1 - e) where k is a pseudo first-order rate constant ((k/K(m))bulletE(o)) and E(o) and S(o) represent total enzyme and substrate concentration, respectively. Pseudo first-order rate constants were directly proportional to enzyme concentrations within the range of enzyme concentration used (5-50 nM); (k/K(m)) was calculated by dividing the pseudo first-order rate constant by enzyme concentration. According to the model shown in , (k/K(m)) should contain information about the interfacial binding as well as catalytic steps. As shown in Fig. 1, the hydrolysis of polymerized mixed liposomes by slower PLA(2) mutants, most notably K56E(Q) and K116E(Q), gradually deviated from the first-order kinetics with the progress of reaction. To obtain rate constants for these mutants that can be compared with those of bovine wild-type and mutant PLA(2)s, we measured the initial velocity of reaction. Assuming that all of the reactions at an early stage follow the scheme shown in , (k/K(m)) can be calculated from the pseudo first-order rate constant ((k/K(m))bulletE(o)) determined from the initial velocity measurement according to ,

where (DeltaF/Deltat)(o) and DeltaF(max) indicate initial velocity described in terms of fluorescence change at 380 nm and the maximal fluorescence change expected from the complete hydrolysis of pyrene-phospholipid, respectively. DeltaF(max) was determined by the addition of an excess amount of bovine PLA(2) to the reaction mixture for a given concentration of phospholipid. Typically, the initial velocity was determined from the fluorescence change in first 2 min, which generally was high enough to allow accurate rate determinations. Within the enzyme concentration range used for K56E(Q) and K116E(Q) (10-100 nM), the initial velocity was linearly proportional to the enzyme concentration and, accordingly, (k/K(m)) values were independent of enzyme concentration. For bovine wild-type PLA(2) and faster mutants including K53E and K10E, the analysis of entire progress curve and the initial velocity measurement yielded essentially the same (k/K(m)) values, which further supported the validity of the latter method (data not shown). Kinetics of PLA(2)-catalyzed hydrolysis of diC(6)PE and diC(6)PG monomers were performed with 0.5 mM phospholipid, 0.16 M NaCl, and 10 mM CaCl(2). The hydrolysis of mixed micelles was measured in the presence of 2 mM Triton X-100, 1 mM sodium deoxycholate, 0.5 mM diC(8)PG, and 10 mM CaCl(2) as described previously (Dua and Cho, 1994). Time courses of the hydrolysis of phospholipids were monitored with a computer-controlled Metrom pH-stat (Brinkmann Instruments, Inc.) in a thermostated vessel. The completion of hydrolysis was confirmed by the addition of an additional amount of enzyme to the mixture after the reaction. Under these condition, the hydrolysis of phospholipids followed first-order kinetics. Thus, (k/K(m)) values were calculated by dividing the pseudo first-order rate constant by enzyme concentration.

Binding Measurements

The binding of PLA(2) to polymerized liposomes was measured fluorometrically as described previously (Wu and Cho, 1994). Typically, the same concentration of protein (2 µM) was added to a solution containing 0.1-100 µM BLPG, and the fluorescence intensity of protein at 345 nm (F) was measured after it reached an equilibrium value. Excitation wavelength was set at 280 nm. To enhance relatively weak fluorescence signal of bovine PLA(2)s, a maximal spectral bandwidth (20 nm) was used for both excitation and emission. Then, the relative fluorescence change (F) for each phospholipid concentration was calculated as (F(max) - F)/(F(max) - F(min)) where F(max) and F(min) represent the protein fluorescence at 345 nm in the absence of BLPG and in the presence of an excess amount of BLPG, respectively. The F values were plotted as a function of total BLPG concentration. Assuming each phospholipid molecule as a ligand that binds independently with apparent dissociation constant K(d) to n equivalent sites on a PLA(2) molecule, values of both n and K(d) were determined by the nonlinear least-squares analysis of the Fversus [PL](o) data using ,

where [E](o) and [PL](0) represent total enzyme and total phospholipid concentrations, respectively.


RESULTS

We designed two groups of bovine pancreatic PLA(2) mutants based on previous structural and mutational analyses of pancreatic enzymes, substrate binding site (in the active site) mutants, and interfacial binding site mutants. Lugtigheid et al. (1993a, 1993b) showed that Arg-53 of porcine PLA(2) (Lys-53 for bovine PLA(2)) is involved in the head-group specificity of the active site. Also, Noel et al.(1991) reported that Lys-56 is involved in the head-group specificity. We therefore generated substrate binding site mutants by reversing (or removing) the positive charge of these lysine side chains; i.e. K53E, K56E, and K56Q. Independently, we have shown (Wu and Cho, 1993; Dua and Cho, 1994; Shen et al., 1994) that cationic residues in the amino-terminal alpha-helix of PLA(2) are involved in its interaction with anionic interfaces. Based on this information and previous structural (Dijkstra et al., 1981) and chemical modification studies (Van der Wiele et al., 1988a, 1988b), we selected Lys-10 and Lys-116 for the generation of interfacial-binding site mutants; i.e. K10E, K116E, and K116Q. SDS-polyacrylamide electrophoresis of mutant proteins showed that they all have an expected molecular mass of 14 kDa (data not shown). To rule out a possibility that mutations caused a gross structural change of protein, we measured the CD spectra of wild-type and mutant proteins. As shown in Fig. 2, all the proteins showed essentially the same spectra, indicating the absence of any major structural change due to mutation. Also, all of the mutants were expressed with comparable efficiencies, which implies that they have similar thermal stability. Lastly, the lack of any deleterious structural change was confirmed by comparable or higher activity of all the mutants toward a monomeric substrate, diC(6)PE (see Table 1).


Figure 2: Circular dichroism spectra of the wild-type (box, 20 µM) and mutant bovine pancreatic PLA(2)s including K10E (bullet, 27 µM), K53E (up triangle, 35 µM), K56E (, 22 µM), K56Q (, 28 µM), K116E (, 30 µM) and K116Q (, 25 µM). Protein concentrations were varied within a narrow range to obtain spectra for a better illustration.





We measured the activity of wild-type and the above mutants toward two different types of polymerized mixed liposomes; pyrene-PE/BLPG and pyrene-PG/BLPG. BLPC-based polymerized mixed liposomes were not used in these studies due to low activity of bovine PLA(2) toward these liposomes (Wu and Cho, 1994). We previously showed (Wu and Cho, 1993) that PLA(2) selectively hydrolyzed pyrene-containing phospholipids inserted in BLPG polymerized liposomes and that the relative activity of individual enzyme species toward different pyrene-containing phospholipids in these polymerized mixed liposomes reflected their genuine head-group preference. Values of (k/K(m)) for all of the enzymes are summarized in Table 1. Based on the finding that bovine enzymes slightly favor as a substrate pyrene-PE over pyrene-PC, (^2)we mainly used pyrene-PE as a zwitterionic substrate. Accordingly, we describe the preference of the wild-type and mutants for anionic phospholipids in terms of the ratio of their activity toward pyrene-PG to that toward pyrene-PE, which will be referred to as PG preference hereafter. If a lysine residue is involved in the substrate binding in the active site, one would expect that its mutation will affect the PG preference of enzyme. If, on the other hand, the lysine residue is involved only in the interfacial binding, its mutation will not change the PG preference but will lower the activity toward all the anionic BLPG based polymerized mixed liposomes.

As reported previously (Wu and Cho, 1994), bovine PLA(2) greatly (15-fold) preferred anionic pyrene-PG to zwitterionic pyrene-PE inserted in BLPG polymerized mixed liposomes. This high PG-preference has been ascribed to the presence of cationic protein residues, most notably Lys-53 and Lys-56, in the active site. Indeed, both the K53E and K56E mutations greatly reduced the preference for the anionic substrate: K53E and K56E favored PG only by 2-3-fold (Table 1). Despite the similar low PG-preference of these two mutants, however, a dramatic difference was found in their catalytic activity toward any BLPG based polymerized mixed liposomes. Toward pyrene-PG/BLPG polymerized mixed liposomes, for instance, K53E was slightly less active than the wild-type enzyme, whereas K56E showed only 0.7% of the wild-type activity. The low activity of K56E was not due to the deactivation of enzyme by mutation, as this mutant was 1.5-fold more active than the wild-type enzyme toward a monomeric substrate, diC(6)PE (Table 1), and retained 50% of wild-type activity toward another monomeric substrate, diC(6)PG (Table 2). These results indicate that both Lys-53 and Lys-56 are involved in the substrate binding in the active site and, more importantly, that Lys-56 is also involved in the interfacial binding. This notion is further supported by kinetic properties of K56Q in which the positive charge of lysyl side chain is neutralized rather than being reversed; it showed not only higher activity than K56E toward pyrene-PE/BLPG polymerized mixed liposomes but also exhibited higher PG-preference.



As expected from their putative role in the binding to anionic interfaces, the mutation of Lys-10 and Lys-116 significantly reduced the activity of enzyme toward anionic BLPG based polymerized mixed liposomes. Toward pyrene-PG/BLPG polymerized mixed liposomes, K10E and K116E showed 20 and 3% of the wild-type activity (Table 2). Again, the low activity of these mutant proteins was not due to the deactivation of enzyme by mutation as they were only slightly less active than the wild-type enzyme toward monomeric substrates, diC(6)PE and diC(6)PG ( Table 1and Table 2). The different degree of rate decrease can be accounted for in terms of the different accessibility of these two lysyl side chains. The crystallographic structure of bovine PLA(2) shows that Lys-116 is fully exposed, but that Lys-10, although located in the same side of protein molecule, is relatively buried among hydrophobic residues in the amino-terminal alpha-helix (Dijkstra et al., 1978, 1981). Furthermore, both K10E and K116E showed the same high PG-preference as the wild-type enzyme. Lastly, K116Q, which was slightly more active toward pyrene-PE/BLPG polymerized mixed liposomes than K116E, showed essentially the same PG preference. Taken together, these results indicate that Lys-10 and Lys-116 are involved only in the interfacial binding and that their mutations do not interfere with the binding of an anionic substrate with the active site.

In order to test if altered kinetic behaviors of bovine PLA(2) mutants toward BLPG based polymerized mixed liposomes were specifically due to the change in their interfacial binding and/or substrate binding, we measured their binding to BLPG polymerized liposomes. We previously showed that one could directly measure the dissociation constant for a PLA(2)-liposome complex fluorometrically using polymerized liposomes, which greatly simplifies the interpretation of kinetic data measured using polymerized mixed liposomes (Wu and Cho, 1994). The binding isotherms of bovine PLA(2) and mutants to BLPG polymerized liposomes are shown in Fig. 3, and thermodynamic parameters determined from the data analysis are summarized in Table 1. The binding isotherms for all the bovine PLA(2)s showed an identical hyperbolic pattern, indicating a lack of any unusual binding mode for a specific mutant. Also, bovine PLA(2) and all of the mutants except K56E have essentially the same n values (30). For K56E, which showed the lowest binding affinity toward BLPG polymerized liposomes, the dissociation constant was calculated assuming n = 30. As shown in Table 2, the relative binding affinity of bovine mutant PLA(2)s for BLPG polymerized liposomes, expressed as a ratio of reciprocal of dissociation constant, is in good agreement with their relative kinetic activity toward pyrene-PG/BLPG polymerized mixed liposomes. For instance, K53E showed only a slight decrease in binding affinity, whereas all of the interfacial binding site mutants exhibited a large degree of decrease. Thus, reduced activity of these interfacial binding site mutants toward BLPG-based polymerized mixed liposomes is mainly due to the decrease in their interfacial binding. In particular, Lys-56, the mutation of which reduced the binding affinity to less than 1% of wild-type binding affinity, appears to be the most critical residue in the interfacial binding of bovine pancreatic PLA(2).


Figure 3: The binding isotherms of the wild-type (circle) and mutant bovine pancreatic PLA(2)s including K10E (up triangle), K53E (), K56E (box), and K116E (bullet). Protein concentrations were 2 µM. See ``Experimental Procedures'' for the definition of F. The solidlines indicate theoretical curves constructed using with n and K values determined from nonlinear least squares analyses.



Lastly, we measured the activity of bovine wild-type and mutant PLA(2)s toward diC(6)PE and diC(6)PG at the concentration below their critical micellar concentration (7 mM) (Wells, 1974). Although the premicellar aggregation of short-chain phospholipids with some snake venom PLA(2)s has been reported (Yuan et al., 1990), it is generally thought that the pancreatic PLA(2)-catalyzed hydrolysis of these substrates below critical micellar concentration does not involve the interfacial binding and that the substrate specificity determined using these substrates reflects the genuine head group specificity of the active site. As summarized in Table 1, the PG-preference determined using monomeric substrates agreed relatively well with that determined using BLPG-based polymerized mixed liposomes. In particular, the interfacial binding site mutants, including K10E, K116E, and K116Q, showed no significant change in activity toward diC(6)PE and diC(6)PG. By contrast, the activity of K53E and K56E increased 7- and 1.5-fold toward diC(6)PE (Table 1), respectively, and decreased 2-fold toward diC(6)PG (Table 2), again indicating their involvement in the substrate binding.


DISCUSSION

In the previous structure-function studies of pancreatic PLA(2)s by site-directed mutagenesis, the activity of mutant proteins was measured using short chain phospholipids either as a monomer or as in micelles. Although these studies provided important insights into structure-function relationships in the actions of PLA(2), they could not readily distinguish the effect of mutation on the interfacial binding from that on the substrate binding in the active site. For instance, Noel et al.(1991) and Lugtigheid et al. (1993a, 1993b) showed that cationic residues at the positions 53 and 56 of pancreatic PLA(2)s are involved in their head group specificity but failed to reveal the participation of Lys-56 in the interfacial binding. In this report, we studied the properties of a large number of bovine pancreatic PLA(2) mutants, including ones previously characterized. As described under ``Results,'' kinetic studies of these mutant proteins using polymerized mixed liposomes and binding studies using polymerized liposomes unambiguously identify key residues involved in the interfacial binding of bovine pancreatic PLA(2) and evaluate their relative contributions. Although we did not determine the tertiary structure of any mutant proteins described herein, their identical CD spectra and comparable activity toward diC(6)PE and/or diC(6)PG preclude a possibility that altered activity of mutants toward polymerized mixed liposomes might result from any deleterious structural changes.

Our kinetic data for K53E mutant are consistent with those reported for R53E of the porcine enzyme (Lugtigheid et al., 1993a), i.e., a large increase in activity toward zwitterionic substrate and a slight decrease toward anionic substrate. This agreement indicates that Lys-53 of the bovine enzyme and Arg-53 of the porcine enzyme play the same role of interacting with ethanolamine or glycerol head group rather than with the phosphate anion. In addition, these data rule out a possibility that Lys-53 of the bovine enzyme is involved in interfacial binding. In an early x-ray crystallographic analysis of bovine pancreatic PLA(2), Dijkstra et al.(1981) proposed that a face of this protein molecule that is directed toward the interface is composed of three sets of residues; one includes the amino-terminal alpha-helix and other two include Lys-56 and Lys-116, respectively. By means of chemical modification studies, Van der Wiele et al. (1988a, 1988b) suggested that Lys-10 and Lys-116 of pancreatic PLA(2)s are involved in the interfacial binding. Our kinetic and binding studies show that these lysines indeed participate in the interfacial binding but that their relative contributions are quite different. Toward pyrene-PG/BLPG polymerized mixed liposomes, the K10E mutation results in a 5-fold rate decrease, whereas the K116E mutation leads to a 30-fold decrease. Also, this rate decrease is in excellent agreement with the decrease in binding affinity for BLPG polymerized liposomes. The decreases in K(d) can be approximately translated into a loss in interfacial electrostatic interactions by 1.0 and 2.0 kcal/mol, respectively, at room temperature using an equation, DeltaDeltaG = -RT ln[K(d) for mutant/K(d) for wild-type], where R and T indicate gas constant and temperature, respectively. These estimated values, which compare well with the known value for electrostatic interactions between two oppositely-charged residues in proteins (1-3 kcal/mol) (Barlow and Thornton, 1983), indicate that Lys-116 makes more significant contributions to the interactions with anionic interfaces than Lys-10 by 1.0 kcal/mol. Most importantly, both kinetic and binding data show that Lys-56 makes larger contributions to the interfacial binding than any other residue studied, especially when the bovine enzyme catalyzes the hydrolysis of anionic interfaces. Toward pyrene-PG/BLPG polymerized mixed liposomes, for instance, the K56E mutation results in a 150-fold rate decrease, whereas the K116E mutation leads to a 30-fold decrease (Table 2). Previously, the K56E mutant of bovine PLA(2) (Noel et al., 1991) and the K56Q mutant of porcine PLA(2) (Lugtigheid et al., 1993b) were reported to have 25 and 67% of the wild-type V(max) value (or k value), respectively, toward micelles of short chain phosphatidyl glycerols. These values are much higher than 0.7 and 4% of residual activity observed for K56E and K56Q mutants of the bovine PLA(2) toward pyrene-PG/BLPG polymerized mixed liposomes. The difference might arise from different physical shapes of micelles and liposomes. Presumably, the binding of the bovine PLA(2) to loosely packed micellar surface might not require strong electrostatic interactions and, consequently, the decrease in electrostatic interactions by the K56E mutation does not cause drastic effects on the enzymatic activity. By contrast, the same mutation exerts dramatic effects on the enzyme activity toward polymerized mixed liposomes because the binding of the bovine PLA(2) to densely-packed large liposome surface depends greatly on electrostatic interactions. To evaluate the effects of mutations on the enzyme activity under a physiologically relevant condition, we measured the activity of bovine wild-type and mutant PLA(2)s using Triton X-100/sodium deoxycholate/diC(8)PG mixed micelles (4:2:1 in mole ratio), the structure of which is relatively similar to that of physiological substrates for pancreatic PLA(2)s, i.e. phospholipids emulsified with bile salts. As summarized in Table 3, the rate decrease for interfacial binding site mutants is, in general, less significant than observed with pyrene-PG/BLPG polymerized mixed liposomes but is much bigger than observed with phosphatidyl glycerol micelles. In particular, the K56E mutant shows only 4% of wild-type activity. Implications from these results are 2-fold. First, Lys-56 is indeed a critical residue in the interfacial catalysis of bovine pancreatic enzyme toward anionic substrates under the physiological condition. Second, our polymerized mixed liposome system serves as a highly sensitive probe to identify essential interfacial binding site groups, which has been difficult with conventional micellar substrates.



The critical involvement of Lys-56 in the interfacial binding of bovine PLA(2) is further supported by two independent studies. First, Tomasselli et al.(1989) showed that Lys-56 was selectively acylated by artificial PLA(2) substrate, 4-nitro-3octanoyloxybenzoic acid (Cho et al., 1988a; Cho and Kezdy, 1991). Earlier, we reported (Cho et al., 1988b) that Lys-7 and Lys-10 of PLA(2) from the venom of Agkistrodon piscivorus piscivorus were selectively acylated by 4-nitro-3-octanoyloxybenzoic acid. We recently showed (Shen et al., 1994) that these two lysines are involved in the binding of the PLA(2) to anionic interfaces and that their selective acylation is due to their proximity to a reactive ester moiety of 4-nitro-3-octanoyloxybenzoic acid, which forms an anionic aggregate. Likewise, one can reason that Lys-56 of pancreatic enzymes is selectively acylated by 4-nitro-3-octanoyloxybenzoic acid because of its critical involvement in the binding to anionic interfaces. Second, Scott et al.(1994) recently reported the electrostatic potentials calculated for a number of secretory PLA(2)s with known tertiary structure. Among these secretory PLA(2), bovine pancreatic PLA(2) showed unique electrostatic potential distribution with high positive potentials around Lys-56. Lys-56 is located in a loop (residues 55-67), which is unique to class I PLA(2), particularly mammalian pancreatic enzymes (Scott and Sigler, 1994). Thus, the critical involvement of Lys-56 in the interfacial binding of pancreatic PLA(2)s implies that these enzymes might have an interfacial binding mode distinct from that of other types of secretory PLA(2)s.

In summary, polymerized mixed liposomes and polymerized liposome provide useful kinetic and thermodynamic systems that allow a systematic structure-function analysis of PLA(2). Studies described herein unambiguously show that Lys-56 of bovine pancreatic PLA(2) is involved in both the interfacial binding and the substrate binding and that its Lys-116 is an essential residue for the interfacial binding. Also, these studies reassess individual roles of Lys-10 and Lys-53. Although not included in this report, the structure-function study for PLA(2) showing considerable activity toward PC liposomes can be further supplemented by measuring the PG-preference of PLA(2) in BLPC polymerized mixed liposomes (Wu and Cho, 1993). Structure-function studies of other types of PLA(2)s based on this approach are in progress.


FOOTNOTES

*
This work was supported by an Arthritis Investigator Award from Arthritis Foundation, a Grant-in-aid from American Heart Association (AHA 92-700), and American Cancer Society Illinois Division). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. .

§
To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-0431.

(^1)
The abbreviations used are: PLA(2), phospholipase A(2); diC(6)PE, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine; diC(6)PG, 1,2-dihexanoyl-sn-glycero-3-phosphoglycerol; pyrene-PE, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine; diC(8)PG, 1,2-dioctanoyl-sn-glycero-3-phosphoglycerol; pyrene-PG, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol; BLPC, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine; BLPG, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol; TPCK, tosylphenylalanyl chloromethyl ketone; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine.

(^2)
R. Dua and W. Cho, unpublished observations.


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