(Received for publication, July 6, 1995; and in revised form, October 5, 1995)
From the
The contribution of a covalent bond to the stability of
complexes of serine proteinases with inhibitors of the serpin family
was evaluated by comparing the affinities of -trypsin and the
catalytic serine-modified derivative,
-anhydrotrypsin, for several
serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that
anhydrotrypsin had little or no ability to compete with trypsin for
binding to
-proteinase inhibitor
(
PI), plasminogen activator inhibitor 1 (PAI-1),
antithrombin (AT), or AT-heparin complex when present at up to a
100-fold molar excess over trypsin. By contrast, equimolar levels of
anhydrotrypsin blocked trypsin binding to non-serpin inhibitors.
Equilibrium binding studies of inhibitor-enzyme interactions monitored
by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed
that the binding of serpins to anhydrotrypsin was undetectable in the
case of
PI or AT (K
>
10
M), of low affinity in the case of
AT-heparin complex (K
7-9
10
M), and of moderate affinity in the case
of PAI-1 (K
2
10
M). This contrasted with the
stoichiometric high affinity binding of the serpins to trypsin as well
as of the non-serpin inhibitors to both trypsin and anhydrotrypsin.
Maximal K
values for serpin-trypsin
interactions of 1 to 8
10
M,
obtained from kinetic analyses of association and dissociation rate
constants, indicated that the affinity of serpins for trypsin was
minimally 4 to 6 orders of magnitude greater than that of
anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the
characteristic fluorescence changes in a P9 Ser
Cys PAI-1
variant labeled with a nitrobenzofuran fluorescent probe (NBD) which
were shown previously to report the serpin conformational change
associated with active enzyme binding. These results demonstrate that a
covalent interaction involving the proteinase catalytic serine
contributes a major fraction of the binding energy to serpin-trypsin
interactions and is essential for inducing the serpin conformational
change involved in the trapping of enzyme in stable complexes.
Protein proteinase inhibitors of the serpin superfamily play
important roles in regulating the serine proteinases of blood
coagulation, fibrinolysis, inflammation, and many other physiologic
processes(1, 2) . These inhibitors are single
polypeptide chain proteins of 400 amino acid residues and are
therefore considerably larger than other non-serpin serine proteinase
inhibitors which contain from 29 to 190
residues(3, 4) . The serpins nevertheless share
certain mechanistic features with non-serpin inhibitor families. Both
serpin and non-serpin inhibitors thus inhibit proteinases by forming
stable equimolar complexes in which a substrate-like interaction is
made between an exposed inhibitor binding loop and the enzyme active
site (1, 2, 3, 4) . However, serpins
differ from non-serpin inhibitors in requiring a large inhibitor
conformational change to trap proteinases in such
complexes(5, 6, 7, 8, 9, 10) .
In this conformational change, the inhibitor binding loop is thought to
collapse from its exposed position on the protein surface and become
inserted into the center of a major
-sheet comprising the core of
the protein. By contrast, in the non-serpin inhibitors, the binding
loop is rigidly fixed in an optimal substrate binding,
``canonical'' conformation capable of tight interaction with
the proteinase with minimal conformational
adjustments(3, 4) .
An additional important difference between the serpins and non-serpin inhibitors is that serpin-proteinase complexes are SDS-stable, whereas non-serpin inhibitor-proteinase complexes are dissociated in SDS. This behavior implies that a covalent interaction stabilizes the serpin complexes, whereas noncovalent interactions stabilize the non-serpin inhibitor complexes(1, 2) . The observation that serpin-proteinase complexes are dissociated by alkaline pH and nucleophiles or spontaneously at a slow rate, producing in all cases reactive-site cleaved inhibitor and active enzyme, has suggested that these complexes are unusually stable tetrahedral or acyl intermediates of an otherwise normal proteolysis reaction(11, 12, 13, 14, 15, 16) . NMR data support such a stabilization of serpin-proteinase complexes as covalent tetrahedral or acyl intermediates(17) . In contrast, the stabilization of non-serpin inhibitor-proteinase complexes predominantly by noncovalent interactions is indicated from the finding that anhydroproteinases, in which the catalytic serine has been converted to dehydroalanine, bind these inhibitors with high affinities similar to those of native proteinases(18, 19) . The absence of a covalent bond between the proteinase active site serine and the inhibitor scissle bond in non-serpin inhibitor-proteinase complexes has been shown clearly from high resolution x-ray crystallographic structures as well as NMR studies of these complexes(4, 20, 21) .
While available evidence supports the idea that serpin-proteinase complexes are stable covalent intermediates linked via the proteinase catalytic serine, the role of this covalent interaction in the inhibitory mechanism and its contribution to high affinity complex formation have not been elucidated. To address these questions, we have compared the affinities of three serpin inhibitors for trypsin and anhydrotrypsin by direct equilibrium binding studies or by kinetic analyses of association and dissociation rate constants for inhibitor-proteinase complex formation. The affinities of two Kunitz inhibitors for native and modified trypsins have also been examined to provide representative examples of non-serpin inhibitors, whose interactions are known not to be significantly affected by the serine modification(18, 19) . Additionally, the involvement of the serpin conformational change in these interactions has been examined with a serpin variant containing a specific fluorescent label which reports this conformational change(22) . Our results reveal that the high affinity interaction of trypsin with the serpin inhibitors and the ability to induce the serpin conformational change is greatly reduced or abolished by the catalytic serine modification in anhydrotrypsin, in marked contrast to the comparable high affinities of trypsin and anhydrotrypsin for the non-serpin inhibitors. These results establish that a covalent interaction involving the proteinase catalytic serine residue is critical for the high affinity interactions of serpin inhibitors with proteinases and for triggering the serpin conformational change.
SBTI (type 1S) and BPTI (from bovine
lung) were purchased from Sigma. Human antithrombin was purified from
outdated plasma by heparin-Sepharose, DEAE-Sepharose, and Sephacryl
S-200 chromatography as described(26) . Human
-proteinase inhibitor was isolated from plasma by
ammonium sulfate precipitation, followed by zinc chelate and
DEAE-Sepharose chromatography(27) . Recombinant PAI-1 (>90%
active) was expressed in Escherichia coli, purified, and
titrated with active site-titrated urokinase as described(28) .
Storage in 5 mM Mes, 0.3 M NaCl, pH 6.0, stabilized
the active form. Latent PAI-1 was made by incubating 0.25
µM active inhibitor in 0.1 M Hepes, 0.1 M NaCl, pH 7.4 buffer at 37 °C for 17 h, after which time
residual inhibitor activity was undetectable (<1%). Variant PAI-1s
with site-directed mutations in the P14 residue (Thr
Arg) and in
the P9 residue (Ser
Cys) were expressed and purified as
described previously(22, 29) . Labeling of the P9 Cys
variant with iodoacetamido-NBD (Molecular Probes) was done as in prior
studies(22) . Reactive site-cleaved antithrombin was prepared
as described(30) . A size-fractionated heparin of M
= 7900 with high affinity for
antithrombin was purified as in previous studies (31) .
Synthetic heparin pentasaccharide corresponding to the antithrombin
binding sequence in heparin (32) was generously provided by Dr.
Maurice Petitou of Sanofi Recherche. Heparin concentrations were
determined by stoichiometric heparin-antithrombin binding titrations
monitored by tryptophan fluorescence enhancement(26) .
Concentrations of native proteins were determined from the 280 nm
absorbance with the use of the following published absorption
coefficients
(litersg
cm
) and
molecular weights: BPTI, 0.83 and 6513(33) ; native and cleaved
antithrombin, 0.65 and 58,000(34) ;
-proteinase inhibitor, 0.48 and 53,000(35) ,
active and latent PAI-1, 1.0 and 43,000(22) . The concentration
of NBD-labeled PAI-1 was determined from the 280 nm absorbance after
correction for the NBD absorbance at this wavelength(22) .
-Trypsin concentrations were determined by active site titration
with fluorescein mono-p-guanidinobenzoate(36) .
Comparison with concentrations determined from the absorbance at 280 nm
and an absorption coefficient of 1.54
liters
g
cm
and
molecular weight of 23,900 (23) indicated 77-86% active
enzyme. Fluorescein mono-p-guanidinobenzoate was used
similarly to determine the active concentration of
-anhydrotrypsin
from the amount of anhydroenzyme necessary to fully liberate trypsin
from its complex with SBTI(24) . Preparations were >80%
active by this assay. SBTI concentrations were determined by titration
of active site-titrated trypsin (10 nM) assuming an equimolar
inhibition stoichiometry(21) . Such concentrations were in
reasonable agreement with those determined from the 280 nm absorbance
with an extinction coefficient calculated from the amino acid sequence (37) .
where F
is the maximal fluorescence
change, [E]
and [P]
are the total enzyme and p-aminobenzamidine
concentrations, respectively, and K
is the
dissociation constant for the enzyme-probe interaction. This equation
assumes a binding stoichiometry of 1:1 as established previously for
the trypsin-probe interaction(40) .
F
and K
were the fitted parameters. Emission
spectra of enzyme-probe complexes and free probe were measured with
excitation at 335 nm and excitation and emission bandwidths of 4 and 2
nm, respectively. After correcting for the appropriate background
spectrum (buffer ± enzyme), spectra of enzyme-probe complexes
were obtained by subtracting the contribution of the free probe
spectrum calculated from the measured K
.
F
in this equation is the observed
fluorescence, corrected for background and dilution, minus the starting
fluorescence, [I]
is the total inhibitor
concentration, K
is the dissociation constant for
the inhibitor-enzyme interaction, and other parameters are as defined
above. This equation follows from the more general cubic equation for
competitive binding under the condition where [P]
[P]
, which was closely approximated in
these titrations ([P]
< 3%)(41) .
where F and F
are
the fluorescence at time t and time 0, respectively, v
is the rate of change in fluorescence at time 0, k
is the first order rate constant for complex
dissociation, [E-I]
is the starting
concentration of complex, and TN is the turnover number for
hydrolysis of substrate by enzyme under the conditions of the
experiment, expressed as the rate of change in fluorescence per unit of
enzyme concentration. The coefficients of the second order polynomial
equation were the fitted parameters, and k
was
calculated from the fitted coefficient of the t
term using the concentration of complex and independently
measured turnover number. The above analysis assumes complex
dissociation is an essentially irreversible process with no significant
contribution due to residual inhibitor association during the initial
rate measurement. Irreversibility of complex dissociation was
demonstrated by showing that the inhibitor is released from the complex
in an inactive, cleaved form rather than in the native intact form (see
``Results''). The contribution of the association process to
the apparent k
measured from the initial rate of
complex dissociation was calculated from the expression,
where [S] is the substrate concentration,
([E] +
[E
S])
/[E -
I]
is the ratio of uninhibited enzyme (free and
complexed with substrate) to inhibited enzyme at time 0, and other
parameters are as defined above. This contribution was <2%,
confirming that it could be neglected.
where v and v
are
the initial substrate hydrolysis rates measured in the absence and
presence of inhibitor, respectively, n is the inhibitor
binding stoichiometry, and the other parameters are as defined above.
The K for the anhydrotrypsin-SBTI interaction
was measured by equilibrium competition with trypsin, whereby
equilibrium was approached either by adding trypsin to
anhydrotrypsin-inhibitor complex or anhydrotrypsin to trypsin-inhibitor
complex(18) . Separate experiments were conducted with 10
nM trypsin, 9 nM SBTI, and either 50, 100, 200, or
500 nM anhydrotrypsin. Residual trypsin activity was measured
after reaching equilibrium by diluting 100 µl of the reaction
mixture into 0.9 ml of 100 µM S-2222 substrate and
measuring the initial rate of substrate hydrolysis at 405 nm.
Attainment of equilibrium was indicated from the indistinguishable
final trypsin activities observed after 16 to 24 h of incubation.
Control incubations in the absence of anhydrotrypsin confirmed the
stability of uninhibited and inhibited enzyme activities over this
time.
Figure 1:
Anhydrotrypsin competition with trypsin
for binding to Kunitz and serpin inhibitors. Inhibitors (10
nM) were preincubated in the absence () or presence of
10 nM (
), 100 nM (
) or 1000 nM (
) anhydrotrypsin (AHTrypsin) prior to initiating
reactions with 10 nM trypsin. Residual enzyme activity was
then measured at the indicated reaction times as described under
``Experimental Procedures.'' Solid lines are fits of
kinetic curves in the absence of anhydrotrypsin by a second order
process.
Figure 2:
Binding of p-aminobenzamidine to
trypsin and anhydrotrypsin. Upper and middle panels,
titrations of the fluorescence enhancement accompanying the binding of p-aminobenzamidine to trypsin (1 µM, 325,
345) or to anhydrotrypsin (10
µM,
335,
355) as
detailed under ``Experimental Procedures.'' The solid
lines are fits by the equilibrium binding equation assuming a 1:1
stoichiometry. Lower panel, emission spectra of p-aminobenzamidine, free or complexed with trypsin or
anhydrotrypsin, obtained with excitation at 335 nm. Concentrations were
100 µMp-aminobenzamidine, 2.5 or 5
µM trypsin, and 5 or 10 µM anhydrotrypsin.
Spectra of complexes were obtained by subtracting the contribution of
the free p-aminobenzamidine spectrum using measured K
values, normalizing to 1 µM complex, and averaging. The free p-aminobenzamidine
spectrum (average of four spectra) was also normalized to 1
µM.
Figure 3:
Binding of Kunitz inhibitors to trypsin
and anhydrotrypsin. Titrations of 0.82 µM trypsin and 10
µMp-aminobenzamidine (upper panel) or
of 5 µM anhydrotrypsin and 100 µMp-aminobenzamidine (lower panel) with BPTI (circles) or SBTI (triangles). Titrations were
monitored from the decrease in relative fluorescence, F/F
(
325
nm,
345 nm), accompanying the displacement of the
probe from the enzyme as the inhibitor is bound, as detailed under
``Experimental Procedures.'' Solid lines are linear
regression fits of titrations with end points calculated as the average
of values obtained after no further fluorescence changes were
detected.
A similar
stoichiometric displacement of p-aminobenzamidine from 1
µM
-trypsin was observed when the enzyme-probe
complex was titrated with the serpin inhibitors,
PI,
antithrombin, antithrombin-heparin complex, and PAI-1. This was again
evidenced from the linear quenching of trypsin-bound p-aminobenzamidine fluorescence by added inhibitor up to an
end point corresponding to the free probe fluorescence and the addition
of
1 mol of inhibitor per mol of enzyme (Fig. 4). As with
the non-serpin inhibitors, stoichiometric binding of the serpin
inhibitors to trypsin was also observed when titrations were conducted
at 10-fold lower enzyme concentrations, implying high affinity
interactions also of the serpin family inhibitors with
-trypsin
characterized by K
values
10
M. By contrast, titrations of 5
µM
-anhydrotrypsin with the same serpin inhibitors
produced no or a comparatively smaller quenching of anhydroenzyme-bound p-aminobenzamidine fluorescence even after several molar
equivalents of inhibitor were added (Fig. 4). Thus, no
significant decreases in probe fluorescence were observed in titrations
with
PI or antithrombin, a modest quenching of probe
fluorescence was produced by titrating with antithrombin-heparin
complex, and a more substantial but less than stoichiometric decrease
in fluorescence occurred when the anhydroenzyme was titrated with
PAI-1. The reduced or undetectable fluorescence changes produced in
titrations of anhydrotrypsin with serpin inhibitors did not appear to
be due to slow binding, since such changes were found to be stable for
at least 3 h in the case of titrations with antithrombin or
antithrombin-heparin complex.
Figure 4:
Binding of serpin inhibitors to trypsin
and anhydrotrypsin. Titrations of 0.82 µM trypsin and 10
µMp-aminobenzamidine (top panel) or of
5 µM anhydrotrypsin and 100 µMp-aminobenzamidine (bottom panel) with
PI (
), AT (
), AT-heparin complex
(
), and PAI-1 (
), as in Fig. 3. AT-heparin complex
was formed by including 3 µM heparin in the enzyme-probe
solution for titration of trypsin and 25 µM heparin for
titration of anhydrotrypsin. Solid lines in the upper
panel are linear regression fits, as in Fig. 3. The solid lines in the lower panel represent the expected
dependence for 1:1 stoichiometric binding.
Titrations of anhydrotrypsin with
antithrombin complexed with full-length or pentasaccharide high
affinity heparins over an extended range of inhibitor concentrations
revealed a more substantial quenching of the bound probe fluorescence.
Heparin or antithrombin alone produced no significant decline in probe
fluorescence, indicating that a specific interaction of the
inhibitor-heparin complex with anhydrotrypsin was responsible for the
fluorescence changes. Addition of a molar excess of SBTI at the end of
these titrations reduced the fluorescence to that of the free probe,
indicating that the fluorescence quenching was due to displacement of
the bound probe from the anhydroenzyme active site. These titrations
were fit well by an equation for competitive equilibrium binding (see
``Experimental Procedures'') (Fig. 5) which indicated
end points indistinguishable from the free probe fluorescence and
similar K values of 6.7 ± 0.5
µM and 9.0 ± 0.3 µM for titrations
with antithrombin-full-length heparin and antithrombin-pentasaccharide
complexes, respectively. Reactive site-cleaved antithrombin in the
absence or presence of saturating heparin (45) produced minimal
displacement of p-aminobenzamidine from anhydrotrypsin,
indicating that an intact reactive center loop was required for
interaction with the anhydroenzyme.
Figure 5:
Binding of native and modified serpins to
anhydrotrypsin. Top panel, titrations of 5 µM anhydrotrypsin and 100 µMp-aminobenzamidine
with antithrombin (), antithrombin complexed with full-length
(
) and pentasaccharide (
) heparins, and reactive
site-cleaved antithrombin with (
) and without (
) complexed
heparin, as in Fig. 4. Antithrombin-heparin complexes were
formed by including heparin in the enzyme-probe solution at 25
µM (native AT) or 100 µM (cleaved
AT)(45) . Relative fluorescence changes are normalized to the
maximum change obtained by addition of a molar excess of SBTI at the
end of the titrations. Bottom panel, titrations of 2.5
µM (
) or 5 µM (
) anhydrotrypsin
with PAI-1, 2.5 µM anhydrotrypsin with P14 Thr
Arg
variant PAI-1 (
) (taken from (29) ), and 2.5
µM anhydrotrypsin with latent PAI-1 (
), all in the
presence of 100 µMp-aminobenzamidine, as in the upper panel. Solid lines in both panels are fits by the
competitive binding equation given under ``Experimental
Procedures.'' Native PAI-1 titrations were globally fit by a
single K
.
Titrations of 2.5 or 5
µM anhydrotrypsin with several molar equivalents of PAI-1
resulted in nearly complete quenching of the bound probe fluorescence.
Global fitting of these titrations showed that a single K of 0.23 ± 0.05 µM for the
PAI-1-anhydroenzyme interaction satisfactorily described the binding
data (Fig. 5). Latent PAI-1, in which the reactive center loop
is inserted into
-sheet A(46) , was considerably less
effective in displacing the probe from the anhydroenzyme. The estimated K
of 7 ± 1 µM for the latent
PAI-1-anhydroenzyme interaction indicated at least a 30-fold reduced
affinity compared to the native inhibitor interaction, suggesting that
optimal anhydrotrypsin binding required the reactive center loop to be
in the native exposed conformation. Together, these results indicated
that non-serpin inhibitors exhibited high affinity interactions with
both trypsin and anhydrotrypsin, whereas serpins showed a high affinity
interaction with just trypsin and a comparatively weak affinity
interaction with anhydrotrypsin.
Figure 6:
Fluorescence changes accompanying the
binding of trypsin and anhydrotrypsin to NBD-labeled P9 Ser Cys
PAI-1 variant. Emission spectra of 0.1 µM NBD-labeled P9
variant alone or after addition of 1 molar eq of trypsin or 16 molar eq
of anhydrotrypsin. See ``Experimental Procedures'' for
experimental details.
Further supporting this conclusion, anhydrotrypsin
bound to a P14 Thr Arg hinge region mutant of PAI-1 which was
defective in reactive center loop insertion(29) , with a K
of 0.04 ± 0.07 µM, fairly
similar to that of active wild-type PAI-1 (Fig. 5). This result
implied that optimal anhydrotrypsin binding does not require loop
insertion. (
)To obtain additional evidence for this
conclusion, the effect of anhydrotrypsin binding to PAI-1 on the rate
of spontaneous insertion of the reactive center loop into
-sheet A
was evaluated from the rate of conversion of active PAI-1 to the
inactive, latent form. The half-life for this inactivation measured at
37 °C (see ``Experimental Procedures'') was increased in
the presence of nearly saturating anhydrotrypsin (1 µM)
from 1.8 ± 0.1 h to 3.2 ± 0.6 h (average of 3 to 5
experiments). Increasing the anhydrotrypsin concentration to 10
µM did not produce any further increase in half-life (2.5
± 0.1 h), indicating that anhydrotrypsin binding antagonizes
rather than promotes the spontaneous insertion of the reactive center
loop into
-sheet A.
Figure 7:
Kinetics of serpin-trypsin complex
dissociation. Dissociation of 1.7 nM serpin-trypsin complexes
in 200 µM tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin
monitored from the acceleration of the hydrolysis of fluorogenic
substrate by the dissociated enzyme as detailed under
``Experimental Procedures.'' Complexes were prepared by
incubating 1 µM enzyme and 5 µM inhibitor
(PI,
), 0.5 µM enzyme and 2.5
µM inhibitor ± 5 µM heparin (AT,
, or AT-H,
) or 0.1 µM enzyme and 0.5
µM inhibitor (PAI-1,
) for 10 min. Solid lines are fits of data (every 10th point is shown) by the parabolic
equation for product formation given under ``Experimental
Procedures'' which reflects a linear rate of enzyme
generation.
Figure 8:
SDS-gel electrophoresis of the products of
serpin-trypsin complex dissociation. A, nonreducing gel of
AT-trypsin complex at time 0 (lane 1) and after 48-h
dissociation (lane 2), reactive-site cleaved AT (lane
3), a mixture of cleaved and intact AT (lane 4), and
intact AT (lane 5); B, reducing gel of PAI-1-trypsin
complex at time 0 (lane 1), after 72-h dissociation (lane
2), dissociated complex mixed with native PAI-1 (lane 3),
and native PAI-1 (lane 4); C, reducing gel of
PI-trypsin complex at time 0 (lane 1), after
72-h dissociation (lane 2), dissociated complex mixed with
native
PI (lane 3), and native
PI (lane 4). D, reducing gel of AT (lanes 1 and 2), PAI-1 (lanes 3 and 4), and
PI (lanes 5 and 6)
either alone (lanes 1, 3, and 5) or after
incubating 1 min with an equal amount (by weight) of anhydrotrypsin. Lane 7 shows anhydrotrypsin alone. Symbols are
enzyme-inhibitor complex (E-I), native inhibitor (I), cleaved
inhibitor (I
), and free enzyme (E) or
anhydroenzyme (E*). The amounts of inhibitor in each lane were
the same for a given gel except for the mixtures where half the amount
was run. Reactive site-cleaved inhibitors were identified based on
their increased mobility in a reducing gel due to loss of a C-terminal
peptide, except for AT whose reactive site-cleaved form was better
resolved from the native form in a nonreducing gel due to a decreased
mobility(30) . See ``Experimental Procedures'' for
other experimental details.
Table 2also compares the affinity of -trypsin and
-anhydrotrypsin for the two non-serpin inhibitors. K
values were determined by equilibrium titrations
of 10
M
-trypsin with BPTI or SBTI,
monitored by the loss of enzyme activity after attainment of
equilibrium. The measured K
values were in fair
agreement with literature values(19, 44) . K
values for anhydrotrypsin-inhibitor interactions
were measured by equilibrium competition of trypsin and anhydrotrypsin
for limiting inhibitor in the case of SBTI (18) or obtained by
assuming the ratio of K
values for trypsin and
anhydrotrypsin interactions previously determined in the case of BPTI (19) . The K
obtained for the
SBTI-anhydrotrypsin interaction agreed well with the previously
reported value(18) . The results shown in Table 2confirm
past studies that the anhydro modification of trypsin produces a
relatively modest reduction in the affinity of Kunitz inhibitor
interactions for the enzyme(18, 19) , as compared to
the substantial affinity reduction of the serpin inhibitor
interactions. Kunitz inhibitor complexes with trypsin or anhydrotrypsin
were completely dissociated upon SDS-gel electrophoresis and showed no
evidence for inhibitor cleavage under the conditions used to dissociate
the serpin-trypsin complexes.
The role of the catalytic serine residue of serine
proteinases in their high affinity interactions with protein inhibitors
of serpin and non-serpin families has been investigated by comparing
the binding of inhibitors of the different families to trypsin and its
catalytic serine-modified derivative, anhydrotrypsin. In agreement with
past studies(18, 19) , the non-serpin inhibitors
examined were found to bind both trypsin and anhydrotrypsin with high
affinities (K = 0.6-230 pM),
confirming that a functional proteinase active site serine residue is
not required to achieve a high affinity interaction. By contrast,
several serpin inhibitors were shown to bind trypsin with high affinity (K
< 12-78 pM), whereas their
binding to anhydrotrypsin was at least 4 to 6 orders of magnitude
weaker (K
= 0.2 µM to >20
µM). Such results indicate that the proteinase catalytic
serine residue is critical for generating a high affinity interaction
with serpin-type inhibitors.
The differences in the affinities of
trypsin and anhydrotrypsin for the two non-serpin inhibitors span the
range of differences in affinities that have been measured for a number
of such inhibitors with native and anhydroenzymes (2- to
130-fold)(18, 19) . These small differences may
reflect a decreased affinity of the P1 inhibitor residue for the S1
site of the anhydroenzyme, as was seen in the case of p-aminobenzamidine binding to this site, due to the inability
of the dehydroalanine residue to engage in the hydrogen bonding and
dipole-dipole interactions of the serine hydroxyl which are necessary
for optimal binding of the P1 residue(19, 47) . ()Such effects may be more severe for Arg than Lys-type P1
residues due to the preference of the trypsin S1 site for Lys over Arg
side chains and could explain the larger effect of the anhydro
modification on SBTI (P1 Arg) than on BPTI (P1 Lys) interactions.
The apparent dissociation constants (K)
determined for the binding of serpins to trypsin from kinetic
measurements of the ratio, k
/k
, represent upper
limits for the true dissociation constants. This is because the
measured k
is the sum of the rate constants for
turnover of the stable serpin-proteinase complex (E-I*) to
generate reactive center cleaved inhibitor (I
) and free
enzyme (E) and for dissociation of the complex back to the
intact inhibitor (I) and free enzyme; i.e. k
= k
+ k
in :
The preferential dissociation of serpin-trypsin complexes to
cleaved rather than intact inhibitor (Fig. 8) resembles the
behavior of other serpin-proteinase complexes (48, 49) and implies that k
k
. k
therefore approximates k
, so that measured K
values,
represented by k
/k
, are much
greater than true K
values, given by k
/k
. The true differences in
affinity of serpins for trypsin and anhydrotrypsin thus greatly exceed
the minimal 4-6 order of magnitude differences observed,
indicating that considerably greater than 40-60% of the binding
free energy of serpin-proteinase interactions is lost when the
catalytic serine is modified (Table 2). Such findings underscore
an important role for the catalytic serine in producing the high
affinity interaction between serpins and their target proteinases, in
marked contrast to the non-serpin inhibitor interactions.
Higher
affinity interactions of serpins with catalytic serine-modified
proteinases other than those measured in the present study have been
reported. These include the interactions of
-proteinase inhibitor with anhydrochymotrypsin (K
50 nM)(50) , of PAI-1
with anhydrourokinase (K
4-6 nM),
and of PAI-1 with a catalytic serine to alanine variant of tissue
plasminogen activator (K
3-5
nM)(51) . High affinity complexes of antiplasmin with
anhydrotrypsin or dichloroisocoumarin-modified chymotrypsin have also
been qualitatively demonstrated(12, 52) . The
observation that certain serpins such as PAI-1 and antiplasmin form
high affinity interactions with catalytic serine-modified proteinases
is in keeping with the demonstrated importance of specific regions
within or outside the reactive center loop of these serpins in their
recognition by target proteinases. Such regions have been shown to
involve specific lysine residues in the carboxyl-terminal end of
antiplasmin which interact with kringle domains of plasmin (53, 54) or residues in the carboxyl-terminal portion
of the PAI-1 reactive center loop which specifically interact with
unique insertion loops in tissue-type and urokinase-type plasminogen
activators(39, 55, 56) . The contribution of
such active site independent interactions to serpin-proteinase complex
formation does not conflict with the substantial active site-dependent
contribution of the catalytic serine residue to the affinity of
serpin-proteinase interactions shown in this study, since none of the
previous studies compared the relative affinities of serpins for native
and active site-modified proteinases. A similar large differential
affinity of PAI-1 and antiplasmin for native and active site-modified
forms of their target enzymes would thus be predicted from our results.
While our findings confirm past observations that noncovalent
interactions can make a significant contribution to the association of
serpins with proteinases, they also show that the contribution of such
noncovalent interactions can vary greatly from serpin to serpin. The
covalent interaction mediated by the active site serine may thus be
sufficient to produce a high affinity association in many cases.
Our
finding that the proteinase catalytic serine residue makes a
substantial contribution to the high affinity interactions of serpins
with their target proteinases is consistent with previous evidence that
serpin-proteinase complexes are stabilized by a covalent bond between
the proteinase catalytic serine and inhibitor P1
residue(11, 12, 13, 17) . The
formation of this covalent bond may enhance the affinity of
serpin-proteinase complexes by triggering the conformational change
thought to be required for stable complex formation in which the
reactive center loop is inserted into -sheet A of the
inhibitor(5, 7, 8, 9, 10) .
This proposal would be in keeping with the observations in this study
that the noncovalent interaction between serpins and anhydrotrypsin
does not appear to induce this conformational change. Thus, the
interaction of a fluorescent-labeled PAI-1 variant with anhydrotrypsin
is not accompanied by the characteristic fluorescence changes which
accompany its interaction with trypsin and which have been shown to
report insertion of the reactive center loop into
-sheet A in
proteinase-complexed, cleaved, or latent forms of PAI-1 (22) .
Moreover, a P14 Thr
Arg PAI-1 variant defective in its ability
to undergo the reactive center loop conformational change (29) bound anhydrotrypsin with an affinity similar to that of
native PAI-1, consistent with the interaction not being dependent on
the conformational change. Additionally, binding of anhydrotrypsin to
PAI-1 diminished the rate of spontaneous loop insertion involved in
converting active PAI-1 to its latent form, indicating that bound
anhydrotrypsin interferes with the serpin conformational change, in
agreement with the stabilizing effects of other catalytic
serine-modified enzymes on the active PAI-1 conformation(57) .
These findings conflict with the suggestion of a previous study that
the interaction of serpins with catalytic serine-modified enzymes
requires the serpin conformational change(52) . This suggestion
was based on the observation that the binding of serpins to
dichloroisocoumarin-inactivated proteinases was abolished when these
serpins were complexed with reactive center loop peptides which block
the conformational change(6, 9) . While the reason for
these discrepant findings is uncertain, the regeneration of active
proteinase from dichloroisocoumarin-inactivated proteinases (58) and efficiency of cleavage of serpin-peptide complexes by
catalytic proteinase (59) could be the basis for the different
findings. In the present studies, the stably inactivated anhydrotrypsin
has clearly been shown to bind PAI-1 without inducing the serpin
conformational change, consistent with the catalytic serine being
required to induce this change.
Insight into how the covalent active site serine interaction and the serpin conformational change stabilize serpin-proteinase complexes comes from a consideration of how Kunitz and other non-serpin inhibitors form stable complexes with proteinases without such a covalent interaction or analogous conformational change. High resolution x-ray crystal structures of complexes of non-serpin inhibitors with serine proteinases have shown that these complexes are stabilized by a complementary substrate-like interaction between an inhibitor binding loop and the enzyme active site(4) . This lock and key interaction results from the inhibitor binding loop being rigidly fixed in an optimal, canonical substrate binding conformation which is common to non-serpin inhibitor families. These inhibitors thus are substrates which utilize most of their available proteinase binding energy to stabilize a Michaelis complex with proteinase, thereby leaving no additional binding energy to stabilize the transition state necessary for proteolysis to proceed beyond the Michaelis complex (60) .
The importance of a covalent interaction and the
serpin conformational change to serpin-proteinase interactions supports
the previously proposed mechanism of serpin action () in
which a substrate-like reaction between inhibitor (I) and enzyme (E) proceeds beyond a Michaelis complex (EI) to
the tetrahedral or acyl intermediate stage of proteolysis (E-I) before the complex is stabilized by the serpin
conformational change (E-I*)(14, 30, 49, 61) .
Implicit in such a suicide substrate mechanism is a competition between
the conformational change which traps proteinase in a covalent
intermediate of the proteolysis pathway and a normal breakdown of the
covalent intermediate along the proteolysis pathway to generate
reactive-site cleaved inhibitor (I
) and free proteinase, as
depicted in .
The existence of such a competing substrate reaction has been amply documented in native and variant serpin reactions(14, 30, 49, 61, 62) . The mechanism of also indicates that the stabilization of serpin-proteinase complexes by the conformational change does not prevent the substrate reaction but only slows it down, consistent with the findings of present and past studies(16, 48, 49) .
There are two
possible mechanisms by which the covalent bond and the serpin
conformational change may stabilize serpin-proteinase complexes.
According to one mechanism, insertion of the reactive center loop into
-sheet A could induce the loop to adopt the canonical conformation
characteristic of non-serpin inhibitors(4, 8) . Such a
mechanism could produce a thermodynamic stabilization of the covalent
intermediate as it does the Michaelis complex in the non-serpin
inhibitors, i.e. by fully utilizing the binding energy of the
reactive center loop interaction with proteinase to stabilize the
ground state of the covalent intermediate, leaving no binding energy
left to stabilize the transition state for further reaction along the
proteolysis pathway. According to the second mechanism, reactive center
loop insertion into
-sheet A may produce a disruption of the
proteinase catalytic machinery which halts further reaction along the
proteolysis pathway(2, 63, 64) . This
disruption could arise from a loss of the optimal alignment of the
catalytic triad, from a dislodging of the reactive bond from its
productive mode of binding in the proteinase active site or from the
loss of contacts between residues flanking the reactive bond and
proteinase subsites. The resulting loss of catalytic power would render
the covalent linkage between the proteinase catalytic serine and
inhibitor P1 residues kinetically stable by greatly reducing the rate
of its breakdown. Such a proposal would be consistent with the rates of
turnover of stable serpin-proteinase complexes of 10
to 10
s
measured in this
and presumably other studies (48, 65, 66) and k
values for cleavage of peptide substrates by
serine proteinases with mutations in the catalytic triad residues of
10
to 10
s
(67, 68) . The observation that
the limiting rates of formation of stable serpin-proteinase complexes
from Michaelis complexes (4-100 s
) (22, 69, 70) also limit the rates of turnover
of substrate forms of these serpins (59) further suggests
10
- to 10
-fold decreases in k
for proteinase cleavage of serpins as a result of stable complex
formation. Similar decreases in k
of
10
-fold result from the disarming of the proteinase
catalytic machinery by mutagenesis(67, 68) .
The main distinguishing features of these two mechanisms is that the former results in a complex thermodynamically stabilized by ground state interactions between the reactive center loop and proteinase active site, whereas the latter produces a complex kinetically stabilized by the slow cleavage of the covalent linkage. Reactive center-loop interactions thus act in this latter mechanism to promote initial Michaelis complex formation and to stabilize the transition state for subsequent covalent complex formation as with a normal substrate and minimally contribute to stabilizing the final serpin-proteinase complex. Initial support for the latter mechanism has come from studies of a natural P1` variant of antithrombin, which showed that the P1` residue stabilizes the transition state leading to the covalent antithrombin-proteinase complex and does not contribute to stabilizing the ground state of this complex(64) . Whether other reactive center loop residues similarly contribute to a transition state rather than ground state stabilization of serpin-proteinase complexes will require further investigations of the sources of the proteinase binding defects in serpin variants with mutations in the reactive center loop.