From the
The energetic contribution of protein solvation-desolvation
reactions to generation of coagulation activated factor X (FXa) by the
extrinsic pathway protease complex was determined using the technique
of osmotic stress. The initial rate of FXa generation by limited
proteolysis of human FX was measured in reaction mixtures with human
tissue factor (TF) and factor VIIa (FVIIa) assembled either in aqueous
phase or on phospholipid membranes. Osmotic stress was induced on the
surface of reacting proteins with either polyethylene glycol, or
dextran of 6000 and 500,000 molecular weight, respectively. These inert
polymers are sterically excluded from the the solvation shells of
proteins and thus increase the water activity in the excluded spaces.
The volume of water transferred either to or from the excluded spaces
during formation of reaction intermediates was calculated from the
ratio of change in free energy of activation with change in osmotic
pressure,
The generation of coagulation FXa
There is little information about local
factors that may regulate functional assembly of TF
Competition for water in biological
microenvironements must favor relatively dehydrated conformations of
enzymes and substrates. Further, the close apposition between proteases
and their substrates required for catalysis is likely associated with
changes in the volumes of water hydrating the interacting protein
surfaces. Thus, it can be expected that a significant component of the
free energy of activation in protein catalysis is work of
hydration/dehydration required in the formation of tight fitting
intermediates and attainment of chemically active configurations
(Colombo et al., 1992; Rand, 1992; Timasheff, 1993; Douzou,
1994). To test this possibility in reactions catalyzed by coagulation
proteases, transfer of water during generation of FXa by the extrinsic
protease complex was examined using the technique of osmotic stress
(OS).
The term ``osmotic stress'' refers to the strictly
controlled removal of water from the surface of a macromolecular
system, (Parsegian, 1986). Osmotic stress is induced experimentally on
the surface of proteins using inert polymers that are excluded from the
proteins' hydration shells (Knoll and Hermans, 1983). The OS
technique has been used and validated extensively to measure
``hydration'' forces influencing short range intermolecular
separations (Parsegian et al., 1986; Prouty et al.,
1985; Rand and Parsegian, 1989; Rau and Parsegian, 1990). The technique
has also been used successfully to measure the hydration volumes
associated with transitions in protein conformation, transitions
between lamellar and non-lamellar phase of membranes (Gawrisch et
al., 1992), permeation of transmembrane channels (Zimmerberg,
1990), DNA-promoter interactions (Robinson and Sligar, 1993), and
enzyme-substrate interactions (Rand et al., 1993).
During measurements, reaction mixtures were stirred
continuously and maintained at a temperature between 32 and 35 °C
in a Reacti-therm heating/stirring module (Pierce). In experiments
designed to examine temperature dependence of reaction rates,
temperatures were varied between 17 and 37 °C using an
electronically controlled thermocycler (Perkin-Elmer).
Reactions
were initiated with the substrate, FX, added to final concentrations
ranging from 0.01 to 3 µM. Reaction mixtures were sampled
at six regular intervals of 0.25-5 min each, and samples were
diluted immediately in 0.2 M EDTA solution to stop the
reaction. Concentration of FXa in each sample was determined as before
(McGee et al., 1992), with chromogenic substrate N-benzoyl-L-isoleucyl-L-glutamyl-L-arginine p-nitroanilide (S222L) purchased from American Diagnostica
(Greenwich, CT). Initial rates of chromogenic substrate hydrolysis were
followed at 405 nm using a microplate reader (V
Observed effects of PGE in reaction rates
were not significantly different in dialyzed as compared with
undialyzed solutions. In contrast, DT solutions contained impurities
that were inhibitory and required extensive dialysis before this
polymer could be used in OS experiments. Dialysis resulting in <10%
dilution of polymer was accomplished using a multichamber dialysis unit
(MRA corporation, Clearwater, FL) at 10 °C with continuous stirring
and back pressure with a column of water of 10
Concentrations of reactants were adjusted as molal rather than molar
to avoid errors resulting from the small but significant volume
occupied by the stressing polymer. At the highest concentrations of PEG
used, this volume corresponded to approximately 10% of the total
solution volume. Osmotic pressure exerted by each polymer concentration
was calculated using published relationships between weight percent of
polymer and osmotic pressure. (Parsegian et al., 1986).
Osmotic pressure of DT solutions was also measured directly using a
membrane osmometer (Wescor, Inc., Logan, UT). Rates of chromogenic
substrate hydrolysis, Spectrozyme FVIIa
(methanesulfonyl-D-cyclohexylalanyl-butyl-arginine-paranitroaniline
monoacetate) (American Diagnostica, Greenwich, CT), at 5
On-line formulae not verified for accuracy This treatment is based on ``rate process theory''
(Glasstone et al., 1941). The observed reaction rate is
proportional to the concentration of a high energy ``activated
intermediate'' that is at equilibrium with reactants. The
proportionality coefficient can be derived independently from general
physical principles. The equilibrium constant expressed in terms of
this coefficient and in terms of the observed reaction rate can then be
subjected to thermodynamic reasoning as any equilibrium constant. In
the resulting relationship, i.e. Equation 1, R is the
gas constant (1.987 cal/mol/degree), h is Plank's
constant (1.584
Differences in
The volume, V, of water transferred during
reactions was calculated as the differential ratio of the free energy
of activation with respect to the osmotic pressure, obtained from the
slope of curves constructed with data from the interval between 0.022
and 0.225 atm, accessible experimentally with both PEG and DT. Reaction
rates at higher osmotic pressures were examined using PEG as the
stressing polymer. The viscosity of DT in solution made preparation of
reaction mixtures containing more than 10% of this polymer difficult.
Curves were generated by either linear or polynomial regression
using the computer program Stat-View 512 (Brain Power, Inc.). This
program calculates regression coefficients by applying the sweeping
operator to the matrix of cross-product deviations. Volumes were
calculated from the linear coefficients using the equivalence: 1 atm
times the volume of one mol of water = 0.430 cal/mol. The effect
of OS on apparent K
Increasing
salt concentration decreased reaction rates measured either with or
without osmotic stress. The free energy of activation increased with
salt concentration and to the same extent under either condition (Fig. 2). The slope of the regression lines fitted to data points
were 5.9 ± 0.5 and 6.2 ± 0.5 kcal/mol/1 M increment in NaCl concentration for stressed and nonstressed
reactions, respectively. These results indicate that the effects of PEG
and NaCl were largely independent of one another.
The rate of reactions assembled on PC:PS
membranes decreased with increasing osmotic stress. The free energy of
activation increased with the osmotic pressure of the polymer solution.
The slope of the regression line fitted to data points (Fig. 4)
was 0.35 ± 0.050 kcal/mol/atm. This corresponded to 814 mol of
H
Osmotic stress induced with inert polymers, either PEG or
dextran, increased the rate of FXa generation by TF
Gibbs free energy of activation decreased with
increasing osmotic pressure at an initial ratio of 2.2 kcal/mol/atm.
This corresponds to 5116 mol of H
The rate of
reactions assembled on phospholipid membranes was less sensitive to
osmotic stress than the rate of reactions in aqueous phase. The effect
of osmotic stress on membrane-assembled reactions was to decrease
rather than to increase the rate. The free energy of activation
increased at a ratio of 0.35 kcal/mol/atm, corresponding to 814 mol of
water/mol of FXa generated. The increase in free energy of activation
suggests that the activated intermediates in membrane reactions undergo
net hydration relative to reactants. Alternatively, the net increase in
the free energy of activation for the reaction on membranes may reflect
two simultaneous but opposite effects of osmotic stress on reaction
rates, mediated by hydration/dehydration reactions of lipid and protein
components, that is the rate acceleration mediated by protein
dehydration could be obscured by concomitant physical changes in the
membrane that may interfere with either assembly of protease complex or
binding of substrate. Aggregation, lipid exchange, and increases in
fluidity of artificial phospholipid vesicles with PEG have been
described (MacDonald, 1985; Massenburg and Lentz, 1993). However, these
physical changes have been observed at PEG concentrations greater than
those found to affect reaction rates in the present studies. The fact
that reactions assembled on PC membranes responded to OS like aqueous
phase reactions suggests that the catalytic role of membranes is
mediated via interaction of factor X with the acidic membrane.
The
change in slope of
The practical applications and biological relevance of examining
macromolecular interactions under osmotic stress has been noted before
(Parsegian et al., 1986; Rand, 1992; Timasheff, 1993; Douzou,
1994). The colloidosmotic pressure (Diem, 1962; Webster, 1982),
generated in plasma by solutes that cannot permeate the vascular
membrane, must also generate osmotic stress on the excluded volumes of
circulating coagulation factors. Similarly, in extravascular spaces,
coagulation factors are subjected to osmotic stress generated by
constitutive biopolymers such as diffusible matrix proteins and
glycosaminoglycans. Changes in either the concentration or physical
distribution of these polymers can conceivably influence
hydration/dehydration reactions involved in the equilibrium between
functional and nonfunctional conformations of coagulation factors. The
extreme sensitivity of the factor Xa generation reaction to osmotic
stress suggests the interesting possibility that changes in the rate of
coagulation reactions may be regulated in vivo by osmotic
pressure changes within the physiologic range of osmotic pressures
measured in plasma (Diem, 1962).
The volumes of water transferred
during formation of enzyme substrate complexes can provide useful data
for protein structure modeling. The volumes of water displaced by
binding of glucose to the active site cleft in hexokinase have been
measured by OS techniques. The information has been used with
space-filling molecular modeling to determine the relative contribution
of surface dehydration versus protein conformation changes
associated with reduction of the cleft upon substrate binding (Rand et al., 1993). The differential sensitivity to osmotic stress
observed between aqueous and fluid phase reactions also has general
relevance to the overall balance of the coagulation system. This
balance is maintained by a series of cross-catalytic reactions
including amplifying and inhibitory loops mediated by both aqueous and
membrane-bound enzymes and inhibitors. Under pathological conditions,
the protein and GAG composition and concentration of interstitial
fluids and plasma may change considerably. For example, necrosis of
atherosclerotic lesions must generate a large concentration of
colloidosmotic active products derived from cellular disintegration.
Subsequent changes in the osmotic and colloidosmotic equilibrium may
disrupt the complex balance of the coagulation cascade. Understanding
the interaction of functional coagulation proteases under osmotic
stress will help in the development of predictive kinetic models for
the behavior of these enzymes in biological environments of reduced
water activity.
The results in this report demonstrate a significant
contribution of water-protein interactions to the energy required for
FXa generation by the extrinsic pathway protease. Results also provide
evidence consistent with a catalytic role of phospholipid surfaces
mediated by facilitation of the dehydration component of the reaction.
In addition, the quantitative information on surface water obtained
should provide a useful complement to x-ray crystallography and
site-directed mutagenesis in the identification of the functional
configuration of coagulation enzymes.
G*/
. For aqueous phase-assembled
reactions,
G* values decreased with
at ratios
of -2.36 ± 0.38 and -2.26 ± 0.26 kcal/mol/atm
for polyethylene glycol and dextran, respectively. These values
correspond to 5488 ± 883 and 5255 ± 604 mol of water
transferred from the reacting protein surfaces per mol of FXa
generated. At a physiologic osmotic pressure of 7 atm the work of
transfer corresponded to 16 kcal/mol, approximately 70% of
G*. The observed osmotic effects were independent of the
viscosity, temperature, and ionic strength of solutions. For reactions
assembled on phospholipid membranes,
G* increased with
at a ratio of 0.35 ± 0.05 kcal/mol/atm, corresponding
to 814 ± 116 mol of water tansferred from bulk solution to
protein surfaces. At physiologic osmotic pressure the work of transfer
is 2.45 kcal/mol, approximately 12% of
G*. Results
indicate that for factor Xa generation in aqueous phase the work of
desolvation is a significant component of the free energy of
activation. Results also suggest that phospholipid membranes catalyze
the reaction by reducing the desolvation component of the free energy
of activation.
(
)by
limited proteolysis of FX via the extrinsic pathway is one of the key
reactions of the blood coagulation cascade. The reaction is catalyzed
under biologically relevant conditions, by a complex formed between
tissue factor (TF), a protein co-factor and activated coagulation
factor VII (FVIIa), a serine esterase. The co-factor has been
identified in many extravascular tissues and is expressed on the
surface of cells as a transmembrane protein (for reviews, see Rapaport
(1991) and Nemerson(1988)).
FVIIa
complexes in vivo. Results from kinetics studies with purified
reagents indicate that purified TF protein either in aqueous solution
or reconstituted into phospholipid vesicles accelerates, by several
orders of magnitude, the rate of FXa generation by FVIIa (Carson and
Konigsberg, 1980). The TF
FVIIa complex assembled on lipid
membranes generates FXa at rates that are approximately 100-fold faster
than rates of the same reaction in aqueous phase. This catalytic effect
of phospholipid membranes suggests that limited exposure of reactants
to solvent water may be important for efficient assembly and function
of coagulation proteases.
Coagulation Proteins
Pure recombinant TF was
purchased from American Diagnostics. This protein was used either in
aqueous solution or reconstituted into mixed phospholipid vesicles,
either 75% phosphatidylcholine, 25% phosphatidylserine, or 100%
phosphatidylcholine in the presence of divalent cations and
deoxycholate, as described previously (Carson and Konigsberg, 1980).
This procedure results in a mixture of small unilamellar and large
multilamellar vesicles, (Slack et al., 1973) and molar ratio
of phospholipid to TF protein of 2.5 10
to 1. Pure
recombinant human FVIIa with specific activity of >2000 units/mg was
a gift from Dr. Ulla Hedner of Nova Nordis (Gentofte, Denmark). Human
plasma factor X with specific activity of 125 units/mg was purchased
from Enzyme Laboratories Inc. (South Bend, IN). These proteins were
electrophoretically homogeneous and functionally pure in chromogenic
and clotting assays.
Measurement of Factor Xa Generation Rates
The rate
of FX conversion into FXa was measured in reaction mixtures prepared in
50 mM Tris-HCl buffer, pH 7.3, containing 0.03-0.4 M NaCl, 5 mM CaCl, and 0.5 mg/ml bovine serum
albumin. Recombinant TF protein was added at 1.5 nM for
reactions assembled in aqueous phase, and lipid-reconstituted TF was
added at 0.02 nM for reactions assembled in lipid phase.
Factor VIIa was added at 1.7 nM for both aqueous and lipid
phase reactions. This concentration of FVIIa is of the same order of
magnitude as FVII concentration in human plasma and sufficient to bind
all the functional TF in both types of reaction mixtures. This was
demonstrated in titration experiments with fixed concentrations of all
reagents, except FVIIa, which was varied from 0.2 to 3.4 nM.
Reaction rates were maximal in reaction mixtures with 1.7 nM FVIIa and did not increase further with higher FVIIa
concentration.
kinetic microplate reader; Molecular Devices, Palo Alto, CA). The
corresponding factor Xa concentrations were calculated from standard
curves calibrated with active site-titrated factor Xa, (Smith, 1973).
Under the conditions of these experiments no FXa generation was
detected in the absence of TF.
Reaction Rates under Conditions of Osmotic
Stress
To examine the effect of osmotic stress on reaction
rates, mixtures included either polyethylene glycol 6000, molecular
biology grade (PEG) (Sigma) or dextran T500 (DT) (Pharmacia Biotech
Inc.) at concentrations ranging from 0 to 10%. The viscosity of PEG and
DT solutions was measured using a calibrated cross arm viscometer
(Internal Research Glassware, Charlotte, NC). Viscosities were directly
proportional to polymer concentration and much higher for DT than for
PEG. Within the range of polymer concentrations used in these studies,
viscosity increased by 0.959 ± 0.08 and 0.473 ± 0.07
centistokes/sec per each 1% (w/w) increase in the concentration of DT
and PGE, respectively.
2.5 cm.
10
M, by TF
FVIIa, at 1.7 nM were measured in Tris-HCl buffer, pH 7.3, 5 mM,
CaCl
, 0.15 M NaCl, containing 0.5 mg/ml bovine
serum albumin and PEG concentrations ranging from 0 to 5% corresponding
to 0-0.384 atm. Rates at 1, 2, 3, 4, and 5% PEG were 9.52
± 0.23, 10.1 ± 0.17, 9.92 ± 0.12, 9.78 ±
0.22, and 9.78 ± 0.11
10
optical
density units/min, respectively, and not significantly different from
the rate (9.88 ± 0.14
10
optical
density units/min) without PEG.
Calculation of Initial Rates of Factor Xa
Generation
Progression curves of FX conversion to FXa were
analyzed using plots of FXa concentration versus reaction
time. Initial steady-state rates were calculated as the slope of
straight lines fitted to experimental data points using computer
routines for regression analyses (Stat-View 512, Brain Power, Inc.).
Initial rates from progression curves that deviated appreciably from
linearity were derived from the initial tangent to the curve calculated
as the second coefficient of a second degree polynomial fitted to the
observed progress curve.
Calculation of Gibbs Free Energy of Activation
The
Gibbs free energy of activation, G*, of FXa generation by
TF
FVIIa was determined from reaction rate coefficients according
to the following equation.
10
cal
s
), k
is Boltzmann's
constant (3.298
10
cal
s
), and the reaction rate, k, is expressed
in s
([FXa]/[TF] s).
G* values, i.e.
G*, were calculated from rates measured in sets
of reaction mixtures containing identical preparations of protein
reactants. Therefore, possible discrepancies between nominal and real
concentration of either TF, FVIIa, or FX would not influence the
accuracy of these differences. For determination of
G*
values at various osmotic pressures, the concentration of substrate was
kept constant and below the apparent K
of
the reaction.
and k
was also examined by measuring initial rates
at increasing concentrations of factor X in reaction mixtures with
0-5.4% PEG. The K
and k
were obtained as the concentration giving
half-maximal reaction rate, and maximal rate divided by enzyme
concentration, respectively. These values were calculated from
rectangular hyperbolas fitted to data by least square minimization
techniques (k
, Biometallics, Inc., Princeton,
NJ)
Effect of Osmotic Stress on the Rate of FX Hydrolysis
by TF
Initial rates of FXa
generation were measured in reaction mixtures containing fixed
concentrations of TF, FVIIa, CaCl, and FX but varied concentrations of
stressing polymers. Osmotic stress was induced with either PEG or DT
included in reaction mixtures at concentrations ranging from 0 to 10%.
The calculated increase in the osmotic pressure of reaction mixtures
induced by the polymers was 0.022-1.3 atm above that of control
reaction mixtures (Fig. 1). Control reaction mixtures were 290
mosm, equivalent to 7 atm, the physiologic osmolarity of plasma at 38
°C (Jeanneret et al., 1954).
FVIIa Assembled in Aqueous Phase
Figure 1:
Change in free energy of activation
with osmotic stress in aqueous phase. Reaction rates were measured at
increasing osmotic pressure in mixtures with 1.5 nM TF, 1.7
nM FVIIa, 5 mM CaCl, 0.15 M NaCl, 0.5 mg/ml BSA, and 80-200 nM FX in Tris
buffer, pH 7.2, at 33 °C. Gibbs free energy of activation
(
G*) was calculated from reaction rate coefficients, k, as
G* = -RT ln kh/k
T (h is
Planck's constant, k
is Boltzmann's
constant, and T is temperature, 306 K). The
G*
values in the ordinate are the arithmetic difference between
G* values of reactions under standard control conditions
and of reactions under an osmotic stress,
, above control.
Osmotic stress was induced with either PEG (A) or with DT (B). The slope of linear regression lines fitted to data
points for the interval between
= 0.022 and
= 0.25 atm was 2.26 ± 0.26 and 2.36 ±
0.38 kcal/mol/atm for reactions with PEG and DT, respectively. The
linear coefficient of a polynomial regression line fitted to all data
points in A was 1.898 kcal/mol/atm. Data are from 12 and 5 experiments
totalling 60 and 30 independent determinations of
G* in A and B, respectively.
Reaction rates increased
with osmotic stress induced with either PEG or DT. The change in free
energy of activation with osmotic pressure calculated using linear
regression for the interval between 0.022 and 0.225 atm, was
-2.36 ± 0.380 and -2.26 ± 0.257 kcal/mol/atm
for reactions with DT and PEG, respectively. A similar value,
-1.898 kcal/mol/atm, was obtained from the linear coefficient of
the curve fitted by polynomial regression to data obtained with PEG for
the interval between 0.066 and 1.3 atm. The average of these values
corresponds to 4597 mol of HO transferred to bulk solution
from the excluded volume/mol of FXa generated by TF
FVIIa. The
calculated work of transfer at 7 atm is 16 kcal/mol. Apparent kinetic
parameters varied with OS (). Assuming that the K
value reflects the equilibrium constant
of factor X-TF
FVII interaction, the change of K
with
corresponds to the
transfer of 1688 ± 400 mol of H
O/mol of FXa
generated.
Independence of the Effects of Osmotic Stress and Ionic
Strength
Reaction rates were measured in mixtures with
concentrations of NaCl ranging from 0.015 to 0.4 M, with and
without osmotic stress. Osmotic stress in these experiments was induced
with PEG at a fixed concentration of 3.1%, corresponding to an increase
in osmotic pressure in the excluded volume of 0.15 atm.
Figure 2:
Electrostatic double layer screening and
osmotic stress. Reaction rates were measured without () and with
(
) 3.1% PEG (0.15 atm) at increasing concentrations of NaCl.
Other components of reaction mixtures were as indicated in legend to
Fig. 1. The slope of regression lines were 5.9 ± 0.5 and 6.2
± 0.5 for reactions with and without PEG,
respectively.
In theory,
assuming that Na and Cl
ions are not
excluded from the protein surfaces, increasing the concentration of
NaCl results both in double layer screening of charges and in osmotic
pressure increases in excluded and nonexcluded volumes. The increase in
osmotic pressure is 5.1 atm/0.1 M increment in NaCl
concentration and should have resulted in an unfavorable change in
G* of 10.2 kcal/mol/0.1 M increment in NaCl
concentration. The much smaller change observed, that is 0.6
kcal/mol/0.1 M increment in NaCl concentration, may perhaps
reflect a favorable change in
G* mediated by
electrostatic effects. Under physiologic conditions, 0.15 M NaCl, the favorable change in
G* would be of some
13.3 kcal/mol and sufficient to compensate for over 80% of dehydration
work. In its simplest interpretation, this result suggests that the
role of univalent salt in the reaction is to overcome electrostatic
repulsion between reactants. Alternatively, NaCl may be excluded from
hydration volumes different from those that exclude PEG. The
electrostatic component of the reaction is currently being examined in
more detail in our laboratory.
Effect of Osmotic Stress at Variable
Temperature
Reaction rates were measured at temperatures ranging
from 17 to 38 °C, both under standard conditions and under an
osmotic stress of 0.323 atm induced with 4.95% PEG. Reaction rates
increased with temperature, and for the interval 17-32 °C the
slopes of Arrhenius-type plots appear linear and not significantly
different for reactions with stressed and nonstressed proteins. (Fig. 3). The activation energy, E,
determined from the slope was 8.24 ± 1.25 and 7.81 ± 0.95
for reactions with and without PEG, respectively. The respective values
of activation enthalpy,
H*, calculated from the
relationship
H* = E
- RT were 7.63 and 7.20 kcal/mol. This result indicates that the
decrease in
G* observed under osmotic stress conditions
is primarily due to a more favorable activation entropy rather than to
a change in rate-limiting step. The value of the activation entropy at
32 °C, calculated from the free energy of activation values,
G*, and the activation enthalpy,
H*,
according to the thermodynamic relationship
G* =
H* - T
S*, was -40.8
and -43.96 cal/mol/degree with and without PEG, respectively.
This corresponds to a favorable change in the entropy of activation of
9 entropy units/atm.
Figure 3:
Temperature and Osmotic Stress. Reaction
rates were measured at temperatures ranging from 17-38 °C,
(290-311 K) in mixtures without () and with (
) 4.95%
PEG (
= 0.324 atm). Other reactants were as indicated
in the legend to Fig. 1. The natural log of the reaction rate
coefficient is plotted against the reciprocal of the temperature in
Kelvin degrees. The slope of the regression lines fitted to data points
are -3.93 ± 0.63 and -4.15 ± 0.47 for
reactions without and with PEG,
respectively.
Effect of Osmotic Stress on the Rate of FX Hydrolysis by
TF
Reaction rates
were measured in mixtures prepared identically as aqueous phase FXa
generation mixtures, except that recombinant TF protein was substituted
by recombinant TF protein reconstituted into phospholipid vesicles.
Concentration of reconstituted TF in (PC:PS) mixtures was 100- and
10-fold lower than TF protein in aqueous phase and PC reactions,
respectively, in order to maintain FXa generation at a similar rate in
each type of mixture.
FVIIa Assembled on Phospholipid Membranes
O transferred from bulk fluid phase to the excluded volume
per mol of FXa generated. In contrast, reaction rates in mixtures with
PC membranes increased with osmotic stress. For the interval between
0.063 and 0.239 atm, the free energy of activation decreased with
at a ratio of -2.035 ± 0.192 kcal/mol/atm. This
corresponds to 4732 ± 446 mol of H
O transferred to
bulk aqueous phase per mol of FXa.
Figure 4:
Change of free energy of activation with
osmotic stress on phospholipid membranes. Reaction rates were measured
at increasing osmotic pressures induced with PEG. Reaction mixtures
contained 0.02 nM TF, reconstituted into phospholipid vesicles
(75% PC, 25% PS). All other reactants were as indicated in the legend
to Fig. 1. The slope of the regression line fitted to data points was
0.35 ± 0.05 kcal/mol/atm, corresponding to 813.9 mol of water
bound/mol of FXa generated.
FVIIa when the
reaction was assembled in aqueous phase. The extent of rate
acceleration was independent of the chemical characteristics of the
stressing polymer, of the viscosity, of the temperature, and of the
ionic strength of the solutions. These results considered together with
results obtained by others in completely different systems (Gawrisch et al., 1992; Rau and Parsegian, 1990; Rand et al.,
1993; Robinson and Sligar, 1994) indicate that the effect observed is
related to the osmotic force induced by the polymers in the excluded
volumes of reacting proteins. The osmotic force that drives water out
of the excluded volume is the net pressure acting on that volume. The
net pressure is a direct function of the difference in water activity
between excluded and nonexcluded volumes. If water is either extruded
or bound during the reaction, the energetic consequences of increasing
the water activity in the excluded volume is reflected in a shift in
the equilibrium position between reactants and products and can be
included as an additive component to Gibbs free energy of activation,
G*.
O/mol of FXa transferred
from the excluded volume during the reaction. At physiologic osmotic
pressures (approximately 7 atm) the work of transfer for this volume of
H
O is 16 kcal/mol, very significant when compared with the
free energy of activation for the reaction calculated at 21 kcal/mol.
These results imply that the functional structure of the reacting
proteins in the activated intermediates up to the rate-limiting step
corresponded to dehydrated conformations relative to those of the
reactants. Further, the large volumes of water displaced suggest that
the extent of interacting surfaces is also large.
G*/
plots observed at
> 0.25 atm in aqueous phase reactions suggests the
existence of at least two different transitions reflected in the
reaction rate. Protein conformation changes required for attainment of
activated intermediates may involve both dehydration of apposing
surfaces and stabilization by hydration of newly exposed protein
regions. Since it is possible for several reaction steps with similar
energy barriers to influence the reaction rate, it is also possible for
hydration/dehydration volumes to vary at each one of these steps.
Table: Change in apparent kinetic parameters with
osmotic stress
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.