From the
To elucidate the role of the P1` residue of the serpin,
antithrombin (AT), in proteinase inhibition, the source of the
functional defect in a natural Ser-394
Antithrombin is the principal protein inhibitor of mammalian
blood coagulation proteinases and a member of the serpin (serine
proteinase inhibitor) superfamily of protein proteinase inhibitors
(Huber & Carrell, 1989; Olson & Björk, 1994). Inhibitors
of this family act by displaying a unique reactive bond in an exposed
loop of the protein, which is recognized by the proteinase as a normal
substrate to be cleaved. Rather than cleaving this bond, however, the
proteinase becomes trapped in a highly stable complex with the
inhibitor, in which the inhibitor-reactive bond is bound tightly at the
active site of the proteinase (Huber and Carrell, 1989; Bode and Huber,
1992; Olson and Björk, 1994). An analogous stable complex appears
to be formed between low molecular weight protein proteinase inhibitors
of other families and their target enzymes (Laskowski and Kato, 1980;
Read and James, 1986; Bode and Huber, 1992). Unique to the serpin
mechanism, however, is that accompanying the formation of the stable
inhibitor-proteinase complex, the inhibitor undergoes a substantial
conformational change in which the exposed reactive center loop is
thought to insert into the major
The reactive bond of
human antithrombin has been identified as the Arg-393-Ser-394
bond, consistent with the specificity of the target proteinases of
antithrombin for cleaving at Arg- X bonds (Björk et
al., 1981, 1982). Characterization of natural and engineered
variants of antithrombin and other serpins have confirmed the
importance of the reactive bond for inhibitor function and in
determining the specificity of the inhibitor for its target
proteinases. Mutation of the arginine residue of this bond, i.e. the P1 residue, to His, Cys, or Pro in natural variants of
antithrombin thus results in a complete loss of inhibitor function
(Erdjument et al., 1988a, 1988b, 1989; Owen et al.,
1988; Lane et al., 1989a, 1989b). Similar consequences result
from mutation of the P1 Arg residue of the related serpins, plasminogen
activator inhibitor-1, and C1inhibitor, to residues other than Lys
(Sherman et al., 1992; Elderling et al., 1992).
Certain P1 mutations of other serpins produce a loss of function with
natural target proteinases but the appearance of function with other
nontarget proteinases (Owen et al., 1983; Derechin et
al., 1990; Rubin et al., 1990), indicating that the P1
residue is a primary determinant of target proteinase specificity.
While such studies have demonstrated a critical role for the P1
residue in serpin inhibitor function, the role of the P1` residue is
less well defined. Thus, mutagenesis of the P1` residue of plasminogen
activator inhibitor-1 has indicated a broad tolerance for different
amino acids in this position (Sherman et al., 1992). In
contrast, mutation of the P1` serine residue of antithrombin to leucine
in the natural variant, antithrombin-Denver, drastically reduces the
inhibitory activity toward thrombin, although it has a less pronounced
effect on the inhibition of Factor Xa (Sambrano et al., 1986;
Stephens et al., 1987; Theunissen et al., 1993).
Further analysis of a number of P1` variants of antithrombin produced
by site-directed mutagenesis has shown that the size and hydrophobicity
of the P1` residue can significantly influence inhibitor function
(Stephens et al., 1988; Theunissen et al., 1993).
Because of the contrasting findings with regard to the importance of
the P1` residue in serpin function, we have further characterized the
functional defect in the natural P1` variant, antithrombin-Denver. The
results of these studies demonstrate the importance of the P1` residue
in determining the specificity of antithrombin for inhibition of
various target proteinases. They additionally establish a mechanism for
the expression of this specificity in which an interaction between the
inhibitor P1` residue and proteinase stabilizes the transition state
and not the final ground state on the pathway to a covalent
antithrombin-proteinase complex. Such findings support a mechanism for
serpin inhibitor function different from that of low molecular weight
protein proteinase inhibitors but similar to that of a suicide
substrate (Fish and Björk, 1979; Olson, 1985; Patston et
al., 1991).
Mutation of the reactive center P1` serine residue to leucine
in the natural variant, antithrombin-Denver, was previously reported to
drastically reduce the rate of inhibition of thrombin in the presence
of the activator, heparin (Sambrano et al., 1986; Stephens
et al., 1987). Antithrombin-Denver retained the ability to
inhibit thrombin as well as Factor Xa at a slow but measurable rate
also in the absence of heparin (Fig. 1). The observed inhibition
was not due to contaminating heparin, since Polybrene had no effect on
the rates (not shown). Fitting the progress curves in Fig. 1by a
first order exponential decay indicated that antithrombin-Denver
inhibited thrombin with a 430-fold reduced pseudo-first order rate
constant ( k
Previous studies have demonstrated that the
reactions of antithrombin with thrombin and Factor Xa occur in two
steps. An initial encounter between inhibitor and proteinase thus
produces a loose, noncovalent complex and is followed by conversion of
this encounter complex to a tight, probably covalent, complex
(Fig. S1) (Olson and Shore, 1982; Craig et al., 1989).
To determine which of the two steps of the inhibitor-proteinase
reaction was affected by the mutation, we measured the dependence of
observed pseudo-first order inhibition rate constants on inhibitor
concentration. Only the heparin-accelerated reactions were examined
because saturation of the encounter complex with normal antithrombin in
the absence of heparin requires millimolar inhibitor concentrations.
The dependence of k
The results of the present study have shown that the P1`
residue of antithrombin functions in determining the specificity of the
inhibitor for its target proteinases and have provided insight into how
this specificity is expressed. The contribution of the P1` residue to
inhibitor specificity is indicated by the variable effects of the P1`
Ser to Leu mutation in antithrombin-Denver on the rates of inhibition
of different target proteinases (). In all cases, the
specificity of the mutant inhibitor is reduced, indicating that all
target proteinases prefer the native inhibitor P1` side chain, but the
extent of this reduction is least for Factor Xa and greatest for
thrombin. As a consequence, the P1` mutation in antithrombin-Denver
results in a specific Factor Xa inhibitor, with Factor Xa being
inhibited 13-130-fold faster than the other proteinases examined
in the absence of heparin and 4-12,000-fold faster in the
presence of heparin. Previous studies aimed at engineering a
recombinant antithrombin with specificity for Factor Xa similarly found
that mutation of the P1` residue of the inhibitor to a bulky
hydrophobic group was sufficient to selectively reduce thrombin
inhibitory activity with only modest effects on anti-Factor Xa
activity. However, the extent of the selectivity achieved was not
quantified in terms of inhibition rate constants, and changes in
inhibitor specificity were only examined with thrombin and Factor Xa in
the presence of heparin (Theunissen et al., 1993). The altered
specificity of antithrombin-Denver for proteinases can be explained by
the different specificities of the S1` substrate binding sites of
proteinases for the mutant P1` residue differentially affecting the
substrate-like recognition of the mutant inhibitor by proteinase. The
observation that the S1` site of thrombin is occluded by a lysine
residue in the x-ray structure of the proteinase has suggested a
restricted specificity of this site for substrate P1` residues (Bode
et al., 1989). Molecular modeling studies have confirmed the
limited accessibility of large hydrophobic side chains to the S1` site
of thrombin (Theunissen et al., 1993), consistent with the
drastic effects of mutating the P1` residue of antithrombin to such
side chains on thrombin inhibitory activity (Stephens et al.,
1988; Theunissen et al., 1993). By contrast, modeling of the
S1` site of Factor Xa has suggested a greater tolerance for
accommodating large side chains, in keeping with the relatively smaller
effects of engineered P1` mutations in antithrombin on Factor Xa
inhibition (Theunissen et al., 1993). The differential effects
of the P1` mutation in antithrombin-Denver on Factor IXa and plasmin
inhibition similarly imply a restricted accessibility of the S1` site
of Factor IXa but a greater permissiveness of the S1` site in plasmin
for the P1` leucine side chain of the variant inhibitor, in agreement
with the more restricted substrate specificity of Factor IXa as
compared with plasmin.
Comparison of the effects of the P1` mutation
in antithrombin-Denver on both heparin-catalyzed and uncatalyzed
reactions with proteinases has shown that the mutation reduces the
second order rate constants for proteinase inhibition to similar
extents in the absence and presence of the polysaccharide. This is
consistent with the reactive center mutation resulting in just a defect
in proteinase recognition and not affecting heparin binding or
conformational activation of the inhibitor. The mechanism of heparin
acceleration of antithrombin-proteinase reactions differs for thrombin
and Factor Xa reactions (Olson et al., 1992). With Factor Xa,
heparin acts mostly as an allosteric activator of antithrombin by
inducing a conformational change in the reactive center region, which
makes the inhibitor more reactive toward the proteinase. With thrombin,
the conformational change does not greatly influence the reactivity of
antithrombin with the proteinase. Instead, heparin acts primarily as a
bridge to promote the binding of thrombin to antithrombin through
adjacent polysaccharide binding sites for the enzyme and inhibitor
(Olson and Björk, 1991). Our findings that heparin binding to
antithrombin-Denver is normal and that the promotion of thrombin
binding to the heparin-bound mutant inhibitor to form an intermediate
ternary complex is normal explain why the heparin accelerating
mechanism for the antithrombin-Denver-thrombin reaction is unaffected.
Likewise, the similar conformational changes induced in mutant and
normal inhibitors, together with the greater tolerance of the S1`
binding site of Factor Xa for binding large hydrophobic side chains,
explain the modest effects of the P1` mutation on the heparin
accelerating mechanism for the antithrombin-Factor Xa reaction. The
previous suggestion that the differential effects of the P1` mutation
on thrombin and Factor Xa inhibition reflect the different mechanisms
of heparin activation is thus not supported by our results (Theunissen
et al., 1993).
While the evidence presented in this and
other studies favors the idea that the P1` mutation in
antithrombin-Denver results in defective proteinase recognition, it was
surprising to find that the mutation did not affect the initial
encounter complex interaction between inhibitor and proteinase but
instead affected the subsequent transformation of the encounter complex
to a stable complex. An additional significant finding was the lack of
an effect of the P1` mutation on the rate of dissociation of the stable
proteinase-inhibitor complex, implying that the mutation does not
influence complex stability. An understanding of these effects is best
appreciated in the context of the suicide substrate mechanism by which
serpins are thought to inhibit their target proteinases ()
(Fish and Björk, 1979; Olson, 1985; Patston et al., 1991;
Hopkins et al., 1993; Schechter et al., 1993; Olson
and Björk, 1994).
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
According to this mechanism, the reaction of inhibitor (I) with
a proteolytic enzyme ( E) proceeds initially as a normal
substrate reaction with the formation of a noncovalent encounter
complex ( E
Our results have shown that the reduced inhibitory activity of
antithrombin-Denver with various proteinases is not due to increased
turnover of the variant along the substrate pathway, as has been
observed with other reactive center variants (Caso et al.,
1991; Austin et al., 1991; Skriver et al., 1991;
Hopkins et al., 1993; Hood et al., 1994; Lawrence
et al., 1994). Mutations in the reactive center loop are
thought to promote a substrate reaction by interfering with the
inhibitor conformational change required for the trapping of
proteinases in stable complexes. While increases in the inhibition
stoichiometry reflecting increased turnover were observed for the
variant inhibitor, these increases were modest relative to the effects
on the inhibition rate constants and suggest relatively small changes
(
Fig. 5
illustrates how
the P1` mutation may affect the rate of formation of the partitioning
intermediate. According to this scheme, the initial encounter complex
formed between antithrombin and proteinase involves a recognition of
just the P1 residue and not the P1` residue. This proposal is
consistent with the primary role of the P1 residue in proteinase
recognition (Owen et al., 1983; Erdjument et al.,
1988a, 1988b; Owen et al., 1988; Lane et al., 1989a,
1989b; Erdjument et al., 1989; Derechin et al., 1990;
Rubin et al., 1990; Sherman et al., 1992; Elderling
et al., 1992) and with rapid kinetic evidence that weak
interactions of antithrombin with the proteinase S1 site are involved
in the formation of encounter complexes (Olson and Shore, 1982; Craig
et al., 1989). The encounter complex is then proposed to be
transformed by a subsequent attack of the catalytic serine of the
proteinase on the inhibitor reactive bond, as with a normal substrate.
This attack induces the P1 carbonyl carbon from a trigonal to a
tetrahedral configuration, which promotes the interaction of the P1`
residue with the S1` site of the proteinase in the transition state
leading to the formation of a tetrahedral intermediate. The formation
of the tetrahedral adduct is envisaged as then triggering the
conformational change, which leads to insertion of the reactive site
loop into the A
In summary,
the results of this study provide additional support that serpins act
as suicide substrates by coupling a normal substrate reaction between
inhibitor and proteinase to an inhibitor conformational change that
arrests the substrate reaction and thereby kinetically traps proteinase
in a stable complex. In keeping with such a mode of action, our results
have shown that the binding energy of the inhibitor P1` interaction
with proteinase is used to lower the activation energy for conversion
of an initial enzyme-inhibitor complex to a covalent tetrahedral or
acyl-intermediate complex, as is typical of a normal substrate. The
proposed mechanism for participation of reactive center residues other
than P1 in the interaction with proteinase through transition state
rather than ground state complementarity may be a general feature of
serpin-proteinase interactions. Further kinetic investigations of the
step or steps affected by such reactive center mutations similar to
those reported in the present study will be required to evaluate this
possibility.
Inhibition stoichiometries
were measured from titrations of proteinase with antithrombin (AT) or
antithrombin-Denver (AT-Denver) in I 0.15 sodium phosphate
buffer, pH 7.4, at 25 °C as in Fig. 2 (see ``Experimental
Procedures''). Average values from two titrations ± range
are reported except for titrations of Factor Xa in the presence of
heparin, in which case the results of a single titration are reported.
We thank Roberta Sheffer, Ann-Marie Frances-Chmura,
Cathy Ruiz, and Rick Swanson for technical assistance and Dr. Peter
Gettins of the University of Illinois-Chicago for critical comments on
the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Leu variant, AT-Denver,
was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin,
and Factor Xa with second order rate constants that were 430-, 120-,
40-, and 7-fold slower, respectively, than those of native AT,
consistent with an altered specificity of the variant inhibitor for its
target proteinases. AT-Denver inhibited thrombin and Factor Xa with
nearly equimolar stoichiometries and formed SDS-stable complexes with
these proteinases, indicating that the diminished inhibitor activity
was not due to an enhanced turnover of the inhibitor as a substrate.
Binding and kinetic studies showed that heparin binding to AT-Denver as
well as heparin accelerations of AT-Denver-proteinase reactions were
normal, consistent with the P1` mutation not affecting the heparin
activation mechanism. Resolution of the two-step reaction of AT-Denver
with thrombin revealed that the majority of the defective function was
localized in the second reaction step and resulted from a 190-fold
decreased rate constant for conversion of a noncovalent
proteinase-inhibitor encounter complex to a stable, covalent complex.
Little or no effects of the mutation on the binding constant for
encounter complex formation or on the rate constant for stable complex
dissociation were evident. These results support a role for the P1`
residue of antithrombin in transition-state stabilization of a
substrate-like attack of the proteinase on the inhibitor-reactive bond
following the formation of a proteinase-inhibitor encounter complex but
prior to the conformational change leading to the trapping of
proteinase in a stable, covalent complex. Such a role indicates that
the P1` residue does not contribute to thermodynamic stabilization of
AT-proteinase complexes and instead favors a kinetic stabilization of
these complexes by a suicide substrate reaction mechanism.
-sheet of the inhibitor. This
conformational change appears to be essential for arresting the
cleavage of the reactive bond initiated by the proteinase and thus for
trapping the enzyme in a stable inhibited complex (Engh et
al., 1990; Carrell et al., 1991; Skriver et al.,
1991; Björk et al., 1992a, 1992b; Björk et
al., 1993; Hopkins et al., 1993).
Materials
Human antithrombin was purified from
outdated plasma by chromatography on heparin-agarose, DEAE-Sepharose,
and Sephacryl S-200 as described previously (Olson, 1988; Olson et
al., 1993). Heparin was a size- and affinity-fractionated
preparation of M7900 (±10%) described
previously (Olson et al., 1992). Human
-thrombin was
generously provided by Dr. John Fenton of the New York State Department
of Health, Albany, NY. Factor Xa, plasmin, and Factor IXa were obtained
from the purified zymogens of these proteinases by activation with
specific enzymes, followed by their purification as described
previously (Björk et al., 1992a). All proteins were
judged pure by SDS gel electrophoresis. Normal and variant antithrombin
concentrations were determined from the absorbance at 280 nm using an
absorption coefficient of 0.65
liters
g
cm
and molecular
weight of 58,000 (Nordenman et al., 1977). Concentrations of
proteinases were determined by active site titration with
4-methylumbelliferyl p-guanidinobenzoate or fluorescein
mono- p-guanidinobenzoate as described previously (Jameson
et al., 1973; Bock et al., 1989). Comparison with the
concentrations determined from the 280 nm absorbance using published
absorption coefficients and molecular weights (Discipio et
al., 1977, 1978; Barlow et al., 1969; Fenton et
al., 1977) showed that all proteinases were >70% active.
Purification of
Antithrombin-Denver
Antithrombin-Denver was purified from the
plasma of a patient heterozygous for the mutation by modification of a
previously described method, which exploits the differential
reactivities of normal and mutant inhibitors with thrombin (Stephens
et al., 1987). Normal and mutant antithrombins were first
copurified by the procedure used to purify normal antithrombin, except
that the Sephacryl S-200 step was omitted. The purified mixture of
normal and abnormal antithrombins was treated at a concentration of
8 µM (
0.5 mg/ml) with 0.55 molar equivalents of
thrombin in the presence of 1 µg/ml Polybrene (Aldrich) at 25
°C. Neutralization of thrombin enzymatic activity was monitored by
chromogenic substrate assay (see below) until the loss of activity had
ceased (
1 h). Residual active thrombin was then quenched with a
2-fold molar excess of
D-Phe-L-Pro-L-Arg-chloromethyl ketone
(Calbiochem). The sample was dialyzed and rechromatographed on
heparin-agarose, which separated the antithrombin-thrombin complex and
chloromethyl ketone-inactivated thrombin from unreacted
antithrombin-Denver. The purified variant antithrombin (10
µM) was subsequently treated again with 0.02 molar
equivalents of thrombin for 3 h to remove any residual traces of normal
antithrombin. The reaction was quenched with chloromethyl ketone as
above, dialyzed, and rechromatographed a third time on heparin-agarose.
The resulting antithrombin was subsequently processed through the
DEAE-Sepharose and Sephacryl S-200 steps as in the purification of
normal antithrombin.
Inhibition Stoichiometry
Fixed levels of thrombin
(5 µM) or Factor Xa (1 µM) were reacted with
increasing levels of antithrombin or antithrombin-Denver ranging from
0.1 to 1.2 mol of inhibitor/mol of enzyme in 20 mM sodium
phosphate, 0.1 M NaCl, 0.1 mM EDTA, 0.1% polyethylene
glycol 8000, I 0.15, pH 7.4, 25 °C. Residual enzyme
activity was assayed after 27 h for thrombin reactions and after 16 h
for Factor Xa reactions. Assays were performed by diluting samples
1000-fold either into 100 µMD-Phe-L-Pip-L-Arg- p-nitroanilide
(S-2238,
(
)
Chromogenix) for thrombin or 200
µM Spectrozyme FXa for Factor Xa, each containing 100
µg/ml Polybrene, and monitoring the initial rate of substrate
hydrolysis from the absorbance increase at 405 nm. Similar titrations
of 0.1 µM thrombin or Factor Xa with normal or variant
antithrombins at molar ratios of inhibitor/enzyme ranging from 0.1 to
1.5 were also conducted in the presence of 0.3 µM heparin
for 3-20 h and assayed for residual enzyme activity as above.
Inhibitor titrations of Factor Xa in the absence of heparin were also
done at the lower enzyme concentration using a 20-70-h
incubation. The inhibition stoichiometry was obtained from the abscissa
intercept of a linear regression fit of measured rates of substrate
hydrolysis versus the molar ratio of inhibitor to enzyme.
SDS Gel Electrophoresis of Antithrombin-Proteinase
Reaction Products
Antithrombin-proteinase complexes were formed
by incubating 6 µM antithrombin or antithrombin-Denver
with 2 µM proteinase. Reactions with thrombin were done in
the presence of 6 µM heparin for 15 min at 25 °C in
0.02 M sodium phosphate buffer, pH 7.4, containing 0.25
M NaCl ( I 0.3), conditions that both avoided the
degradation of the inhibitor-proteinase complex by free proteinase that
occurs in the absence of polysaccharide and minimized the
salt-dependent substrate reaction of the inhibitor that occurs in the
presence of heparin (Olson, 1985). Factor Xa reactions were done in the
absence of heparin for 1 h at 25 °C in I 0.15, pH 7.4
sodium phosphate buffer. Reactions containing 25 µl were quenched
with an equal volume of SDS sample buffer, boiled for 2 min, and then
electrophoresed under nonreducing conditions in 10% polyacrylamide gels
using the Laemmli buffer system (Laemmli, 1970). Proteins bands were
visualized by Coomassie Blue R-250 staining.
Kinetics of Inhibitor-Proteinase Complex
Formation
Reactions of antithrombin or antithrombin-Denver with
proteinases in the absence or presence of heparin were done in total
reaction volumes of 40 or 50 µl in polystyrene cuvettes. The buffer
was 20 mM sodium phosphate, 0.10 M NaCl, 0.1
mM EDTA, 0.1% polyethylene glycol 8000, and 1 mg/ml bovine
serum albumin, pH 7.40, at 25 °C (except for Factor IXa reactions,
where bovine serum albumin was omitted, and EDTA was present at 1
mM). Plasmin reactions were done in the presence of 20
mM -aminocaproic acid to stabilize the enzyme against
autolysis. Reactions were conducted under pseudo-first order conditions
by employing at least a 10-fold molar excess of antithrombin or
antithrombin-heparin complex over proteinase. A molar excess of
antithrombin over heparin was also used to fully saturate (>90%) the
polysaccharide chains and minimize antagonism of the reaction by free
heparin (Olson, 1988). Measured equilibrium binding constants were used
to calculate concentrations of antithrombin-heparin complex. Proteinase
concentrations were 0.1-100 nM for thrombin, 10-20
nM for Factor Xa, 10-100 nM for plasmin, and
200 nM for Factor IXa. Heparin-accelerated reactions of normal
antithrombin with thrombin and Factor Xa were done in the presence of
2-5 mM p-aminobenzamidine to slow reactions
sufficiently for manual sampling. Reaction rates were corrected for the
effect of the competitive inhibitor as described previously (Olson and
Björk, 1991; Olson et al., 1992). The addition of bovine
serum albumin served to minimize proteinase adsorption and was found
not to significantly affect the rates of proteinase inhibition. For
antithrombin-Factor IXa reactions, 1 nM hirudin (Sigma) was
included to neutralize a 0.006% thrombin contamination detectable from
the hirudin-dependent loss in enzymatic activity observed with the
fluorogenic substrate assay for Factor IXa activity. Reactions were
quenched at various times with 0.8-1.0 ml 50 µM
Tosyl-Gly-Pro-Arg-7-amido-4-methyl coumarin (Sigma) (thrombin and
Factor IXa), 100 µM S-2238 (thrombin), 200 µM
Spectrozyme FXa (American Diagnostica) (Factor Xa), or 200
µMD-Val-L-Leu-L-Lys- p-nitroanilide
(Chromogenix) (plasmin), all containing 100 µg/ml Polybrene.
Residual enzyme activity was measured from the initial rate of
substrate hydrolysis, either from the increase in fluorescence at
380 nm,
440 nm for thrombin and
Factor IXa reactions, or from the absorbance increase at 405 nm for
thrombin, Factor Xa, and plasmin reactions. Control incubations in the
absence of inhibitor were used to establish the rate corresponding to
fully active enzyme, as well as the stability of the activity over the
time course of the reactions. Observed pseudo-first order rate
constants ( k
) were obtained from the slopes of
semilog plots of enzyme activity versus time or by nonlinear
regression analysis of data by a single exponential decay function
(Olson et al., 1993). Second order rate constants were
obtained by linear regression analysis of plots of k
versus the antithrombin or antithrombin-heparin complex
concentrations, after correction in the latter instance for the free
inhibitor reaction (Olson, 1988).
Kinetics of Inhibitor-Proteinase Complex
Dissociation
Antithrombin-proteinase complex dissociation rate
constants were measured essentially by the method of Jesty (1979).
Complexes were prepared by incubating proteinase (0.5 µM
thrombin or 1 µM Factor Xa) with 5 µM
antithrombin and 5 µM heparin at 37 °C in I 0.15 sodium phosphate buffer for 10 min. Dissociation was then
induced by diluting 5-27 µl of the incubation mixture into
1.0 ml (final volume) chromogenic substrate consisting of 400
µM S-2238 or 400 µM Spectrozyme FXa plus 100
µg/ml Polybrene (final concentrations) in I 0.15 sodium
phosphate buffer at 37 °C. Complex concentrations after dilution
ranged between 2.5 and 27 nM. The initial rate of complex
dissociation (<0.5% dissociation) was continuously monitored for 1 h
at 37 °C from the parabolic increase in absorbance at 405 nm due to
the reappearance of enzyme activity. The absorbance changes were fit by
a second order polynomial, and the dissociation rate constant was
obtained from the fitted parameters using the measured turnover number
at 37 °C and the total concentration of complex as described
previously (Danielsson and Björk, 1983; Olson et al.,
1993). Measured rate constants were not appreciably affected by
changing the antithrombin or proteinase concentrations, by including
heparin to accelerate complex formation, or by increasing the substrate
concentration employed to monitor complex dissociation, consistent with
the measured rate constant not containing a significant contribution
from the association process. Reported time-dependent decreases in the
dissociation rate constant due to complex aggregation were minimized by
the short reaction time of 10 min employed for complex formation
(Danielsson and Björk, 1983).
Heparin Binding Titrations
Binding of high
affinity heparin to antithrombin or antithrombin-Denver was measured by
fluorescence titrations in which the enhancement in protein tryptophan
fluorescence accompanying polysaccharide binding was used to monitor
the interaction (Olson and Shore, 1981; Olson et al., 1993).
Titrations were done with 50 nM inhibitor in 20 mM
sodium phosphate, 0.10 M NaCl, 0.1 mM EDTA, 0.1%
polyethylene glycol 8000, pH 7.4. Fluorescence measurements were made
at 280 nm,
340 nm on an SLM 8000
spectrofluorometer. Relative fluorescence changes,
F/ F
=
( F
F
)/ F
(where
F
and F
are
observed and starting fluorescence values, respectively), were computer
fit by the quadratic equation describing the equilibrium binding
process from which values of the dissociation constant, stoichiometry,
and maximum fluorescence change were obtained (Olson et al.,
1993).
) as compared with antithrombin. By
contrast, the variant antithrombin inhibited Factor Xa with only a
6.5-fold reduced k
as compared to the normal
inhibitor. Reactions of antithrombin-Denver with thrombin were
characterized by monophasic inactivation curves and indistinguishable
second order rate constants when inhibitor/proteinase ratios were
varied from 20,000 to as low as 20, indicating that the slow
inactivation of this proteinase by the variant inhibitor was not due to
contaminating wild-type inhibitor. The greatly different reductions in
the rates of proteinase inactivation by antithrombin-Denver relative to
antithrombin for thrombin and Factor Xa provided additional evidence
that the inactivation of these proteinases by the variant inhibitor was
not the result of contaminating normal antithrombin.
Figure 1:
Reactions of antithrombin and
antithrombin-Denver with proteinases in the absence of heparin.
Reactions of 5 µM inhibitor with 10 nM thrombin
(,
), 1 nM thrombin (
), or 20 nM
Factor Xa (
,
) were conducted at I 0.15, pH 7.4,
25 °C as described under ``Experimental Procedures.'' In
control incubations, the inhibitor was omitted (
). Solid lines are computer fits to a single exponential decay
function.
The reactions
of antithrombin-Denver and wild-type antithrombin with both thrombin
and Factor Xa required approximately equimolar levels of inhibitor and
proteinase for complete inhibition (Fig. 2, ). These
findings indicated that the observed inactivation of proteinases by
antithrombinDenver was due to the formation of 1:1 stoichiometric
complexes like those formed in normal antithrombin-proteinase
reactions. Heparin increased the inhibition stoichiometries of both
normal and variant inhibitors with the two proteinases (),
due to the polysaccharide promoting a competing reaction of
antithrombin with the enzymes as a substrate (Olson, 1985; Olson et
al., 1992). Somewhat greater inhibition stoichiometries were
observed for the variant as compared with the normal inhibitor both in
the absence and presence of polysaccharide, suggesting the P1` mutation
increased the amount of inhibitor reacting as a substrate. However,
such effects were small relative to those on the rate of proteinase
inhibition. SDS gel electrophoresis of mixtures of thrombin or Factor
Xa with a molar excess of normal or mutant inhibitors confirmed that
equimolar enzyme-inhibitor complexes were the primary reaction products
for both normal and variant inhibitor-proteinase reactions
(Fig. 2).
Figure 2:
Stoichiometry and products of normal and
variant antithrombin-proteinase reactions. A,
titrations of fixed levels of thrombin (5 µM) or
Factor Xa (1 µM) with antithrombin () or
antithrombin-Denver (
). Experimental details are given under
``Experimental Procedures.'' Solid lines are linear regression fits from which the reaction stoichiometry
was obtained from the abscissa intercept. B, SDS gels of the
products of the reactions of antithrombin or antithrombin-Denver with
thrombin ( left gel) and with Factor Xa ( right gel). Conditions are detailed under ``Experimental
Procedures.'' Lane 1, 2.3 µg of thrombin or
1.8 µg of Factor Xa; lanes 2 and 3, 8.7
µg of antithrombin and antithrombin-Denver; lanes 4 and 5, 8.7 µg of normal and variant antithrombins
reacted with 2.3 µg of thrombin or 1.8 µg of Factor Xa. Protein
standards with molecular weights of 97,000, 67,000, 45,000, 30,000, and
20,000 are included on the left- hand side of each
gel.
The equilibrium binding of high-affinity heparin
( M 7900) to antithrombin-Denver or
antithrombin was compared by titrations of the 40% protein fluorescence
enhancement that accompanies this binding (Fig. 3). The averaged
results from two titrations gave K
values
(± range) of 12 ± 1 and 16 ± 5 nM,
stoichiometries of 0.9 ± 0.1 and 1.0 ± 0.1 mol of
heparin/mol of inhibitor, and maximum relative fluorescence changes of
40 ± 1 and 36 ± 2% for antithrombin and
antithrombin-Denver, respectively. The similar binding affinities and
fluorescence enhancements indicated that heparin binding to the mutant
inhibitor, and the conformational change induced by this binding, were
largely unaffected by the mutation.
Figure 3:
Equilibrium binding of heparin to
antithrombin and antithrombin-Denver. Antithrombin () or
antithrombin-Denver (
) (50 nM) was titrated with the
indicated molar ratios of heparin to inhibitor at I 0.15, pH
7.4, 25 °C and heparin binding monitored from the relative change
in protein fluorescence,
F/ F, as described under
``Experimental Procedures.'' Solid lines are nonlinear regression fits to the quadratic equation for ligand
binding to a protein with equivalent, noninteracting
sites.
Second order rate constants were
measured for the reaction of antithrombin-Denver with a number of
target proteinases under pseudo-first order conditions in the absence
and presence of heparin levels sufficient to saturate the inhibitor
(). The results showed that the effect of the
antithrombin-Denver mutation strongly depended on the proteinase
inhibited, although for each proteinase the effect was similar in the
absence or presence of heparin. Thus, the reduction in the inhibition
rate constant was greatest for the thrombin reaction (430-fold without
heparin and 310-fold with heparin), somewhat less for the Factor IXa
reaction (120-fold without heparin and 240-fold with heparin),
significantly less for the plasmin reaction (40-fold without heparin
and 28-fold with heparin), and least for the Factor Xa reaction
(6.8-fold without heparin and 3.8-fold with heparin). As a consequence
of the similar reductions in second order rate constants for
heparin-catalyzed and uncatalyzed reactions, heparin enhanced the rate
of all antithrombin-Denver-proteinase reactions to an extent similar to
that of normal antithrombin. Of note is the substantially lower second
order rate constant observed for the heparin-dependent inhibition of
Factor IXa by plasma antithrombin in this study as compared with
previous work (Jordan et al., 1980). Direct monitoring of the
formation of antithrombin-Factor IXa complexes by SDS gel
electrophoresis and densitometric analysis confirmed the validity of
the inhibition rate constant measured by loss of activity. The extent
of heparin acceleration of this reaction may thus be much smaller than
previously reported.
on antithrombin-heparin
complex concentration for the reactions of antithrombin-Denver with
thrombin and Factor Xa is shown in Fig. 4. Saturation of
k
occurred in an experimentally accessible range
of inhibitor-heparin complex concentration for the thrombin reaction
but not for the Factor Xa reaction. The linear dependence of
k
found for the latter reaction indicated that
only a second order inhibition rate constant could be obtained (Table
II). Nonlinear least squares analysis of the hyperbolic
saturation curve for the thrombin reaction yielded values of 0.23
± 0.02 µM for the dissociation constant for the
ternary encounter complex interaction and 0.027 ± 0.001
s
for the first order rate constant for conversion
of the encounter complex to a stable complex (Fig. S1).
Comparison with values previously determined for the reaction of normal
antithrombin-heparin complex with thrombin, using the same heparin
fraction (Olson and Björk, 1991), clearly revealed that the P1`
mutation results in a minor, 1.9-fold increased dissociation constant
for the initial encounter complex interaction but a substantial
190-fold reduction in the rate constant for conversion of the encounter
complex to a stable complex (Fig. S1). The effect of the P1`
mutation is thus nearly all in the second, covalent reaction step.
Figure S1:
Scheme 1.
Figure 4:
Inhibitor concentration dependence of
pseudo-first order rate constants for reactions of antithrombin-heparin
complex with proteinases. Rate constants were measured as detailed
under ``Experimental Procedures.'' Solid lines are computer fits of data by a rectangular hyperbola or a straight
line.
To determine whether the mutation affected the stability of the
final antithrombin-proteinase complex, we examined the kinetics of
dissociation of the complexes with thrombin or Factor Xa. This was done
by forming complex with a molar excess of inhibitor over proteinase (to
minimize complex degradation by free proteinase) and then extensively
diluting the complex into a substrate solution and following the
regeneration of proteinase activity. Measurements were made at 37
°C to increase the rate of the dissociation reaction.
Indistinguishable rate constants of (8.0 ± 0.7)
10
s
and (7.6 ± 1.0)
10
s
were found for the
dissociation of complexes of thrombin with antithrombin or
antithrombin-Denver, respectively. Equivalent rate constants (within
experimental error) of (6.8 ± 0.6)
10
s
and (7.3 ± 1.6)
10
s
were also measured for the
dissociation of Factor Xa complexes with antithrombin and
antithrombin-Denver, respectively. These results indicate similar
stabilities of antithrombin and antithrombin-Denver complexes with
proteinases.
I) in which the inhibitor reactive bond is
bound at the enzyme active site. This is followed by an attack of the
catalytic serine of the enzyme on the inhibitor reactive bond to
produce a covalent tetrahedral or acyl intermediate complex
( E-I). The fate of the intermediate complex is governed by the
relative rates of continued reaction along the substrate pathway to
release cleaved inhibitor (I
) and active
proteinase ( E) or of the arrest of the substrate reaction by
an inhibitor conformational change, which traps the proteinase in a
stable complex ( E-I*). The stable complex can subsequently
dissociate to cleaved inhibitor and active proteinase, but at an
extremely slow rate (Jesty, 1979; Danielsson and Björk, 1983).
3-fold) in the ratio of rate constants for partitioning of the
intermediate E-I complex along substrate or inhibition
pathways (Hood et al., 1994). The predominant effect of the
P1` mutation is therefore not to interfere with the inhibitor
conformational change in which the reactive center loop is thought to
insert into
-sheet A, consistent with the localization of the the
P1` residue far from the inserted ``hinge'' region of the
loop (Skriver et al., 1991; Carrell et al., 1991;
Björk et al., 1992a, 1992b; Hopkins et al.,
1993; Björk et al., 1993; Hood et al., 1994;
Lawrence et al., 1994). Since the P1` mutation in
antithrombin-Denver affects the rate of conversion of the encounter
complex to stable complex but does not significantly affect the
reaction of the inhibitor as a substrate or the rate of stable complex
dissociation, it follows that the mutation primarily affects the rate
of formation of the common intermediate, E-I, which precedes
the partitioning step ().
-sheet of the inhibitor and the consequent
trapping of proteinase at the tetrahedral intermediate stage of the
substrate pathway. This trapping is proposed to disrupt the P1`
interaction and thereby explains why the P1` mutation does not affect
the rate of complex dissociation. An alternative mechanism in which
trapping of the proteinase occurs at the acyl-intermediate stage of
reactive bond cleavage is also possible.
Figure 5:
Proposed model for participation of the
antithrombin P1` residue in the inhibition of proteinases. According to
the model, reaction of antithrombin ( I) with a proteolytic
enzyme ( E) proceeds initially like a normal substrate reaction
by the formation of an encounter complex in which the inhibitor P1
residue interacts with the enzyme S1 site. Attack of the catalytic
serine residue of the enzyme on the inhibitor reactive bond
subsequently induces the P1` residue to interact with the enzyme S1`
site in the transition state leading to the tetrahedral intermediate
complex. Proton transfer from the Ser to His and Asp residues of the
catalytic triad occurs concomitant with this attack. Formation of the
tetrahedral intermediate then triggers the inhibitor conformational
change, which arrests the substrate reaction and kinetically traps the
enzyme in the tetrahedral intermediate complex. Completion of the
substrate reaction competes with this conformational trapping,
resulting in proteolytically cleaved inhibitor and free
enzyme.
According to the proposed
mechanism of Fig. 5, the binding energy of the P1` residue
interaction with proteinase is used to stabilize the transition state
complex leading to the tetrahedral intermediate and not the final
ground state complex; i.e. the P1` residue functions to lower
the activation energy and thereby to increase the rate of formation of
the tetrahedral intermediate, but it does not contribute to the
stability of the trapped intermediate. This mechanism thus accounts for
the primary effect of the P1` Ser to Leu substitution in
antithrombin-Denver on the rate of conversion of the encounter complex
to a stable complex and the absence of an effect on the rate of
dissociation of the stable complex. Such a mechanism contrasts with
that of low molecular weight proteinase inhibitors, which interact
noncovalently with their target proteinases through an extended contact
region typically involving the P6-P3` residues of the reactive
center loop and the proteinase active site (Laskowski and Kato, 1980;
Read and James, 1986; Bode and Huber, 1992). Diagnostic of the
thermodynamic stabilization, which characterizes the latter
inhibitor-proteinase complexes, is the primary effect of alterations of
the reactive center loop residues on dissociation rather than on
association rate constants for complex formation (Empie and Laskowski,
1982). Our observation that the P1` mutation in antithrombin-Denver
does not affect the rate constant for dissociation of the stable
complex but only the rate constant for complex formation thus argues
against a similar thermodynamic stabilization of
antithrombin-proteinase complexes by such an extended complementary
interaction. Our results therefore do not support proposals that
insertion of the serpin reactive center loop into -sheet A induces
an active ``canonical'' conformation in the loop like that of
low molecular weight proteinase inhibitors which forms an extended
contact interaction with proteinase that is responsible for stabilizing
the inhibitor-enzyme complex (Carrell et al., 1991; Bode and
Huber, 1992; Hopkins et al., 1993). Our findings instead argue
that the stability of the final complex arises from a kinetic rather
than thermodynamic trapping mechanism as is characteristic of the
suicide substrate mechanism of Fig. 5. The kinetic trapping may
involve the reactive center loop conformational change disrupting the
interactions of the inhibitor reactive bond with the catalytic residues
of the proteinase in the tetrahedral intermediate; e.g. by
displacing the P1 carbonyl oxygen from the oxyanion hole of the enzyme.
The resulting loss of enzyme catalysis could account for the slow
dissociation of the covalently linked inhibitor-proteinase complex to
cleaved inhibitor and free proteinase (Danielsson and Björk, 1983)
and thus explain the trapping of proteinase in a stable complex.
Moreover, the kinetically stabilized complex would not require any
additional interactions other than the covalent interaction linking
inhibitor and proteinase to account for its stability.
Table: Inhibition stoichiometries for
antithrombin-proteinase reactions
Table: 0p4in
Value
obtained using a single inhibitor concentration.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.