(Received for publication, September 28, 1994; and in revised form, November 2, 1994 )
From the
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 (PLA
). Based on
previous structural and mutational studies, several bovine PLA
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
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
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
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.
Phospholipase A (PLA
; (
)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
-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
has an
interfacial binding site separated from the active site (for a recent
review, see Scott and Sigler(1994)). Recent structural analyses of
PLA
-inhibitor complexes for some secretory PLA
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
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
catalysis made it difficult to clearly delineate
structure-function relationships from kinetic analyses using
conventional PLA
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
and ones interacting with the
interfacial binding site of PLA
(Wu and Cho, 1993, 1994).
In this report, we present kinetic and binding studies of bovine
pancreatic PLA
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.
Figure 1:
The hydrolysis of
pyrene-PG (0.5 µM)/BLPG (9.5 µM) polymerized
mixed liposomes by bovine wild-type PLA (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
. 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 and P
, and (k
/K
)
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
(1 - e
) where k is a pseudo
first-order rate constant
((k
/K
)
E
)
and E
and S
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
)
was
calculated by dividing the pseudo first-order rate constant by enzyme
concentration. According to the model shown in , (k
/K
)
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
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
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
)
can be
calculated from the pseudo first-order rate constant
((k
/K
)
E
)
determined from the initial velocity measurement according to ,
where (F/
t)
and
F
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.
F
was determined by the
addition of an excess amount of bovine PLA
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
)
values
were independent of enzyme concentration. For bovine wild-type
PLA
and faster mutants including K53E and K10E, the
analysis of entire progress curve and the initial velocity measurement
yielded essentially the same (k
/K
)
values,
which further supported the validity of the latter method (data not
shown). Kinetics of PLA
-catalyzed hydrolysis of
diC
PE and diC
PG monomers were performed with
0.5 mM phospholipid, 0.16 M NaCl, and 10 mM CaCl
. The hydrolysis of mixed micelles was measured in
the presence of 2 mM Triton X-100, 1 mM sodium
deoxycholate, 0.5 mM diC
PG, and 10 mM CaCl
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
)
values
were calculated by dividing the pseudo first-order rate constant by
enzyme concentration.
where [E] and
[PL]
represent total enzyme and total
phospholipid concentrations, respectively.
We designed two groups of bovine pancreatic PLA 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
(Lys-53 for bovine PLA
) 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
-helix of PLA
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
PE (see Table 1).
Figure 2:
Circular dichroism spectra of the
wild-type (, 20 µM) and mutant bovine pancreatic
PLA
s including K10E (
, 27 µM), K53E
(
, 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 toward these liposomes
(Wu and Cho, 1994). We previously showed (Wu and Cho, 1993) that
PLA
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
)
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, (
)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 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
PE (Table 1), and retained 50% of
wild-type activity toward another monomeric substrate,
diC
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, diCPE and diC
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
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
-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 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
-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
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
s showed an identical
hyperbolic pattern, indicating a lack of any unusual binding mode for a
specific mutant. Also, bovine PLA
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
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
.
Figure 3:
The binding isotherms of the wild-type
() and mutant bovine pancreatic PLA
s including K10E
(
), K53E (
), K56E (
), and K116E (
). 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 PLAs toward
diC
PE and diC
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
s has been reported
(Yuan et al., 1990), it is generally thought that the
pancreatic PLA
-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
PE and
diC
PG. By contrast, the activity of K53E and K56E increased
7- and 1.5-fold toward diC
PE (Table 1), respectively,
and decreased
2-fold toward diC
PG (Table 2),
again indicating their involvement in the substrate binding.
In the previous structure-function studies of pancreatic
PLAs 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
, 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
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
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
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
PE
and/or diC
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, 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
-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
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
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,
G = -RT ln[K
for mutant/K
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
(Noel et al., 1991) and
the K56Q mutant of porcine PLA
(Lugtigheid et al.,
1993b) were reported to have
25 and 67% of the wild-type V
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
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
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
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
s using Triton X-100/sodium
deoxycholate/diC
PG mixed micelles (4:2:1 in mole ratio),
the structure of which is relatively similar to that of physiological
substrates for pancreatic PLA
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 is further supported by two independent studies.
First, Tomasselli et al.(1989) showed that Lys-56 was
selectively acylated by artificial PLA
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
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
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
s with
known tertiary structure. Among these secretory PLA
, bovine
pancreatic PLA
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
, particularly mammalian pancreatic enzymes (Scott and
Sigler, 1994). Thus, the critical involvement of Lys-56 in the
interfacial binding of pancreatic PLA
s implies that these
enzymes might have an interfacial binding mode distinct from that of
other types of secretory PLA
s.
In summary, polymerized
mixed liposomes and polymerized liposome provide useful kinetic and
thermodynamic systems that allow a systematic structure-function
analysis of PLA. Studies described herein unambiguously
show that Lys-56 of bovine pancreatic PLA
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
showing considerable activity toward PC liposomes can be further
supplemented by measuring the PG-preference of PLA
in BLPC
polymerized mixed liposomes (Wu and Cho, 1993). Structure-function
studies of other types of PLA
s based on this approach are
in progress.