molecule adheres to a membrane substrate, and the motions of
the enzyme and phospholipid molecules in the bilayer or mixed
micelle allow hydrolysis of many molecules before the enzyme
leaves the membrane or becomes inactive.13 A result of the
scooting mode of activity is that the enzyme shows a preference
to hydrolyzing phospholipid molecules at an organized interface
over molecules in free solution.15 It has been found that the
enzyme first binds to a membrane substrate and then draws a
phospholipid molecule into the catalytic site, as two distinct steps.16
Knowledge of the structure of different PLA2s and observation of
kinetics via AFM,17 TEM,18 fluorescence,19 and titration tech-
niques20 have supported this hypothesis.
serves a twofold purpose. First, it maximizes interaction between
the enzyme and substrate. Second, it allows the PLA2 to pull a
phospholipid molecule out of the plane of the phospholipid bilayer
in order to reach the active site for hydrolysis,25 reducing
interactions between the hydrophobic paraffin chains and water
when the phosphatidylcholine molecule is displaced upward from
the bilayer. The area covered by the enzyme is equivalent to ∼30
phospholipid molecules, where each phospholipid occupies an
area of ∼0.6 nm2 on the membrane surface.26,27 The number of
binding sites available to the enzyme can be easily estimated with
this information.
Cobra (Naja naja naja) venom and human group X phospho-
lipase A2 exhibit stronger binding to zwitterionic than to anionic
membranes.24,28-31 In the present work, N. naja naja phospholi-
pase A2 is introduced into unilamellar liposome samples prepared
from a zwitterionic phospholipid, dimyristoylphosphatidylcholine
(DMPC), having saturated fatty acid tails that are each 14 carbon
atoms long. The gel to liquid-crystalline phase transition temper-
ature of DMPC occurs near 23.5 °C and over a temperature range
of ∼1 °C. At the phase transition temperature of the lipid,
segregated domains of gel and liquid crystal rafts exist simulta-
neously,32 and this coexistence leads to regions of disorder
between the phases. Pure phospholipid bilayers have been shown
to be poor substrates at temperatures outside the phase transition
region15 due to the lack of structural defects that are starting points
for PLA2-catalyzed hydrolysis. It is currently thought that these
regions of membrane disorder between phase domains are where
phospholipase A2 activity begins.15 The lag in enzyme activity has
been observed and attributed to accumulation of hydrolysis
products, specifically the lysophospholipid, that perturb the
bilayer.32,33 Experiments have shown that there appears to be a
threshold concentration of products that allows the hydrolysis
reaction to proceed rapidly in a “burst phase”,33,34 providing there
is sufficient phase segregation disorder for initial hydrolysis to
occur. Previous measurements of the kinetics of phospholipase
A2 have been successful in obtaining kinetic parameters for activity
on populations of lipid micelles16,35,36 and bilayers.37-39 Previous
studies of DMPC hydrolysis by N. naja naja PLA2 have used lipid
micelles (with Triton-X surfactant molecules) as substrates35,36
where the curvature of the structures reduces the interactions
between the hydrophobic lipid tails, analogous to defects in a lipid
bilayer.
For a rigorous kinetic description, it is important that adhesion
to the lipid bilayer is regarded as a separate step. The overall
scheme can be described using the model of Deems et al.16 as
E + A h EA
EA + B h EAB
EAB f EA + P
(1)
(2)
(3)
where E is the enzyme, A is a binding site on the membrane
surface, B is a phospholipid molecule, and P represents the
products of the reaction. The first step of the scheme, eq 1, is
simply the adsorption of the enzyme to the membrane. This
equilibrium is described by the dissociation constant,16 Kd
)
[EA]/[E][A], and is reportedly achieved within 1 s.21 The second
step of the kinetic scheme, eq 2, describes the binding of
molecules into the active site of the enzyme. The third step of
the kinetic scheme, eq 3, describes reaction of substrate molecules
to form a product with a rate constant, k3.
The concentration of enzyme in solution is simple to determine,
but the structure of the enzyme and the nature of its binding to
a membrane must be known to accurately estimate the num-
ber of binding sites on a vesicle, [A], and thus [EAB]. The
structures of many different PLA2 enzymes have been solved by
X-ray crystallography.22 The PLA2 isoenzymes are relatively small
(11-15 kDa) proteins, and their amino acid sequences and folding
are responsible for their lipid membrane specificity.23,24 When a
phospholipase A2 enzyme binds to a substrate, it covers an area
of 17.5 nm2 and excludes water from the space between the
enzyme and the substrate molecules.25 The exclusion of water
(14) Berg, O. G.; Gleb, M. H.; Tsai, M.-D.; Jain, M. K. Chem. Rev. 2001, 101,
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de Haas, G. H. Biochim. Biophys. Acta 1982, 688, 341-348.
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Mittler-Neher, S.; Spinke, J. Chem. Phys. Lipids 1991, 57, 363-374.
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(28) Ghomashchi, F.; Lin, Y.; Hixon, M. S.; Yu, B.-Z.; Annand, R.; Jain, M. K.;
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