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INTRODUCTION |
1-Antichymotrypsin
(ACT)1 (I) a serine
proteinase inhibitor (serpin) 398 amino acids long (1), is an acute
phase protein in human plasma that participates in regulating the
inflammatory response (2); forms a complex with prostate-specific
antigen, a serine proteinase marker for prostate cancer (3); and is a
major component of amyloid plaques in the brains of Alzheimer's disease patients (4, 5), possibly forming a complex with amyloid
peptide (6). As is typical of serpins (7, 8), ACT forms an
enzymatically inactive, covalent complex (designated E*I*) with its
target proteinases, most likely corresponding to the acyl enzyme
(9-12) that resembles the normal intermediate in substrate turnover.
Both E and I undergo substantial conformational changes in forming
E*I*, which releases free enzyme (E) and cleaved ACT (I*) only very
slowly (13, 14). The nonlability of E*I* has been attributed either to
distortion of the enzyme active site within the complex (14-17) or to
inaccessibility of the covalent E-I linkage toward attacking
nucleophilic water, or both.
Cleavage of I to form released I* occurs between residues 358 and 359 of ACT within the so-called "reactive center loop" (RCL), which in
intact ACT extends out from the rest of the molecule, contains a
segment of modified
-helix (18) and is the primary interaction site
between the inhibitor and the target proteinase. Following standard
nomenclature (19), residues 358 and 359 are designated P1 and P1',
respectively. The RCL extends from approximately P17 to P9'. In
released I*, residues P1-P14 are inserted into
-sheet A, the
dominant structural element in ACT, as strand 4A (s4A). The C-terminal
portion of I, designated the postcomplex fragment (residues 359-398),
is retained in a noncovalent complex with the remainder of I, in which
the P1 and P1' residues are separated by 70 Å (20). Fluorescence
energy transfer studies suggest substantial or even complete s4A
insertion within E*I* for several serpin:serine proteinase pairs (7,
21-23), but this point remains uncertain because in no case has an
E*I* structure been solved.
Whereas full inhibition of
-chymotrypsin (Chtr) requires only 1 equivalent of WT-ACT (i.e. the interaction is characterized by a stoichiometry of inhibition (SI) of 1), ACT inhibition of another
target proteinase, chymase, has an SI of 4 at pH 8 (24), and even with
Chtr, some mutations within the reactive center loop yield inhibitors
with SI > 1 (3). These results, which parallel results obtained
with other serpins, indicate a partitioning between substrate and
inhibitor pathways when serpins interact with serine proteinases, in a
mechanism akin to that of a suicide substrate (8, 13, 25).
Recently, we proposed a kinetic mechanism (Scheme I) for the formation
of E*I* from ACT and Chtr, based largely on rapid kinetic studies of
the interactions of Chtr with both WT-rACT and with a fluorescent
derivative of a P7 variant of rACT,
(7-methoxycoumaryl-4)-methyl-A352C-rACT (11, 26). In this scheme, which
provides a general, albeit minimal, description of the serpin-serine
proteinase interaction, (a) formation of EI' from the
encounter complex E·I is at least partly rate-determining in overall
E*I* formation; (b) the P1-P1' linkage is preserved in EI';
(c) formation of EI' involves substantial RCL insertion into
-sheet A, which therefore occurs prior to acyl enzyme formation; and
(d) partitioning between substrate and inhibitor pathways
occurs prior to E*I* formation. Evidence for an intermediate between
E·I and E*I* has also been obtained for the interaction of
-1-protease inhibitor with Chtr (27) and the interaction of
plasminogen activator inhibitor with plasminogen activator (28).
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Here, in order to explore further the mechanism of E*I* formation,
we extend our studies by investigating the interaction with Chtr of two
derivatives of rACT in which a fluorescent group, 7-[4-(aminosulfonyl)-2,1,3,benzoxadiazole)] (ABD) has been placed at
residues P11 and P13 within the RCL. These residues are of special
interest because they fall within the so-called "hinge" region of
the RCL (residues P10-P14) that plays a key role in the partitioning
between the substrate and inhibitor pathways. Thus, as recently
reviewed (7, 29), mutations at residues P14 and P12, particularly those
in which Arg or Pro replaces a smaller residue, such as Ala or Ser,
convert serpin inhibitors into substrates, presumably because they
affect the rate at which the RCL inserts into
-sheet A during E*I*
formation. In addition, annealing the peptide corresponding to residues
P8-P14 to intact
1-protease inhibitor converts this serpin from an
inhibitor of trypsin to a trypsin substrate by preventing RCL insertion
(30).
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine Chtr was obtained from Calbiochem. Enzyme
concentration was estimated using A280 nm equal
to 2.1 ml/mg/cm, giving values consistent with those determined by the
hydrolysis rate against a standard substrate (1). The chromophoric
proteinase substrate SucAAPF-p-nitroanilide, bovine serum
albumin, dithiothreitol, and phenylmethylsulfonyl fluoride were
obtained from Sigma. Standard proteins for SDS-PAGE calibration were
from Bio-Rad. HPLC-grade acetonitrile was from Fisher, and HPLC-grade
trifluoroacetic acid was from Pierce (sequanal grade). DNA primers for
PCR reactions were synthesized at the Nucleic Acid Facility, University
of Pennsylvania Cancer Center.
Construction, Expression, and Purification of T345C-rACT,
E346C-rACT, and S348C-rACT--
The three variants were constructed
using sequence overlap expression polymerase chain reaction (31, 32)
and the ACT expression vector described previously (1, 33). Polymerase
chain reaction products, representing the entire coding region, were
cut with BstXI, gel-purified, and inserted in the correct
reading orientation in pZMS. Mutation sites were verified by DNA
sequencing. Variant proteins were purified to homogeneity as described
earlier (1, 34) except that in the second chromatography step,
SE-Sepharose replaced double-stranded DNA cellulose. This
chromatography was carried out at pH 6.5 (10 mM MES) using
a NaCl gradient. Variant proteins eluted at 0.4 M NaCl.
Their concentrations were estimated by A280 nm,
using a value of 0.86 mg/ml/cm (35), giving values consistent with
those determined by Bradford analysis (30).
Preparation of ABD Derivatives--
A typical labeling reaction
mixture contained 20 µM of variant protein, 200 µM dithiothreitol, and 1 mM ABD-F (Wako
Bioproducts) (4-(aminosulfonyl)-7-fluoro-2,1,3, benzoxadiazole, added
from a stock solution of 20 mM ABD-F in DMF) in 50 mM Tris-HCl, 50 mM KCl, pH 8.3. The reaction
was allowed to proceed in the dark at 4 °C for 16 h and stopped
by adding 20 mM dithiothreitol. Unreacted probe was removed
by exhaustive exchange with 20 mM NaPi, pH 7.0, buffer in a
Centriprep-30 (Amicon).
Labeling stoichiometry was determined spectrophotometrically by
absorption measurements at 280 nm and 384 nm, using the following extinction coefficients: for ACT,
280 36,000 (26) and
384 1,100 M
1 cm
1
(this work); for bound ABD (25),
280 3,000 and
384 7,800 M
1
cm
1. To correct for light scattering, observed
absorbances at 500 nm were subtracted from values at other wavelengths.
This correction was always
8% of measured values at 384 nm. Control
experiments showed negligible reaction with the unique Cys residue in
WT-rACT, Cys-237, which is buried in the hydrophobic core of the protein.
The above procedure gave virtually complete (
95%) derivatization of
E346C-rACT and S348C-rACT, forming ABD-E346C-rACT and ABD-S348C-rACT,
respectively. On the other hand, T345C-rACT was much more resistant to
derivatization, and ABD-T345C-rACT was not prepared.
Preparation of HNE-cleaved ABD-E346C-rACT--
Cleaved
ABD-E346C-rACT was formed by incubation of human neutrophil elastase
and intact ABD-E346C-rACT (18 µg) at a molar ratio of 1:40 in a final
volume of 0.7 ml containing 20 mM NaPi, pH 7.5, at room
temperature for 6 h. Reaction was stopped by addition of
N-methoxysuccinyl-AAPV-chloromethylketone (Bachem) to a
final concentration of 15 µM. Verification that cleavage
was complete was provided by SDS-PAGE analysis.
Determination of SI--
SI values for ACT variants and
derivatives were determined either by inhibition of Chtr activity, as
described (26), using the chromophoric substrate
Suc-AAPF-p-nitroanilide, or by SDS-PAGE analysis. In the
latter approach, reaction mixtures containing equal volumes (10 µl)
of Chtr (varying concentration) and of 4 µM (final
concentration) WT-rACT, variant rACT, or an ABD derivative of an rACT
variant in 20 mM NaPi (pH 7.0) were incubated at 25 °C
for 15 min. Reactions were stopped by adding SDS nonreducing loading
buffer. The samples were then subjected to SDS-PAGE analysis (see
below), which separates intact ACT from both cleaved ACT and ACT
complexed with Chtr. Values of SI were determined from the extrapolated
concentration of Chtr just sufficient to completely remove the band due
to intact ACT (Fig. 1).
Determination of Second-order Rate Constants for Inhibition of
Chtr--
Rate constants (ki) for inhibition of
Chtr were measured at 25 °C under second-order conditions. The
reaction mixture contained Chtr (5 nM) and the inhibitor at
a concentration of SI × 5 nM in 100 mM
Tris-HCl, pH 8.3, 0.005% Triton X-100 (1). The reaction was quenched
at various times by adding 20 µl of 10 mM
Suc-AAPF-p-nitroanilide in 90% Me2SO into 1 ml
of reaction mixture, and residual Chtr activity was determined. Plots
of (enzyme activity)
1 versus time were linear,
with slopes equal to ki.
Fluorescence Measurements--
Stopped-flow fluorescence
emission spectra were acquired using an Applied Photophysics
stopped-flow spectrofluorometer with excitation at 384 nm and detection
at 480 nm.
Rapid Quenched-flow--
Rapid quenched-flow kinetic studies
were carried out using a KinTek Chemical-Quench-Flow model RQF-3 as
previously (11). Reactions were quenched with 0.1 N HCl and
analyzed by either SDS-PAGE or reverse phase-HPLC.
SDS-PAGE Analysis--
Analyses were performed on quenched
samples as previously (1), using 12% polyacrylamide gels. Aliquots
containing 2-5 µg of total protein were precipitated with an equal
volume of 20% trichloroacetic acid for 30 min. The wet pellet was
taken up in 20 µl of nonreducing sample buffer (0.031 M
Tris-HCl (pH 6.8), 1% SDS, 5% glycerol, and 0.001% bromphenol blue),
except as otherwise indicated. Small amounts of Tris base were added to
neutralize any remaining trichloroacetic acid, and phenylmethylsulfonyl
fluoride was added to a final concentration of 2 mM to
rapidly inactivate any renatured Chtr. Samples were boiled for 4 min
prior to electrophoresis. Gels were stained with Coomassie Blue and
destained with 7.5% AcOH and 5% MeOH. Gel scanning was performed on a
ScanMaker (9600XL) from Microtek, using Adobe Photoshop, over a
concentration range for which band intensity is proportional to the
amount of protein present. Gel images were processed using the National
Institutes of Health Image program (version 1.61). The reproducibility
of band intensities at specific time points was within ±5%.
HPLC Analysis--
Quantification of postcomplex fragment,
corresponding to amino acids 359-398, was performed by reverse
phase-HPLC analysis, as described (2).
Data Analysis--
Stopped-flow spectrophotometric data were fit
using Pro-Kineticist global analysis and simulation software, as
described by the manufacturer (Applied Photophysics). Typically, five
traces were averaged with data smoothing. Quenched-flow data were fit using Hopkinsim (version 1.7) software from Johns Hopkins University. In fitting results to Scheme III, stopped-flow data gave values for
k1, k2, and
k3 (assuming that the fluorescence of
EIc is essentially identical to that of E*I*). Values of
k4 were obtained by fits of quenched-flow data,
using values for k1, k2,
and k3 derived from stopped-flow analysis.
Attempts to fit data allowing reactions in Schemes II or III to be
reversible gave values of reverse rate constants that were highly
variable, although always very low compared with the forward rate
constants. Hence, the reactions were treated as irreversible. Fits of
rate constant data to Scheme IV were performed with Igor Pro 3.13.
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RESULTS |
SIs and Second-order Rate Constants--
All three variants
(T345C-rACT (P14), E346C-rACT (P13), and S348C-rACT (P11)), as well as
the two fluorescent derivatives (ABD-E346C-rACT and ABD-S348C-rACT),
form SDS-stable complexes with Chtr. T345C-rACT has an SI value of 3.8 as measured by titration of Chtr activity (Table
I). The other four variants or
derivatives have SIs just over 1.0, as measured either by titration of
Chtr activity or by SDS-PAGE analysis, measuring the decrease in
intensity of the band due to intact inhibitor as a function of the
Chtr/inhibitor ratio (Fig. 1; see also
Fig. 4). The second-order rate constants (ki) for
inhibition of Chtr by ABD-E346C-rACT and ABD-S348C-rACT are virtually
identical to the rate constant for inhibition by WT-rACT (Table I),
paralleling the lack of effect of amino acid substitution at residues
P11 (38) and P13 (39) on inhibitory activity of other serpins. These
results are in accord with the expectation that mutations of
"P-odd" residues in the P10-P14 hinge region should not affect
inhibitory activity, because, on insertion into
-sheet A, P-odd side
chains point into solution (40).

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Fig. 1.
SI determination by PAGE analysis. The
intensity of the intact inhibitor band (arbitrary units) seen on
SDS-PAGE analysis (see Fig. 4) of reaction mixtures of Chtr and I (4 µM) is plotted against
[Chtr]t/[I]t in the mixtures. Incubation
was for 15 min at 25 °C. Buffers employed were 20 mM
NaPi (pH 6.0 and 7.0) and 20 mM acetate (pH 5). Gel
scanning was performed as described under "Experimental
Procedures." The apparent x-intercept value of 1.11 at pH
7.0, corresponding to an SI of 0.90, was adjusted to give an SI of 1.1 in Table I, based on the minor formation of cleaved I (see under
"Results"), also detected by PAGE.
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Fluorescence Emission Spectra--
Excitation of a solution of
ABD-E346C-rACT at 384 nm gives the emission spectrum shown in Fig.
2, with
max equal to 502 nm. Addition of a small excess of ABD-E346C-rACT to Chtr leads to formation of the E*I* complex and a large increase in fluorescence intensity accompanied by a slight blue shift (1-2 nm), consistent with
a shift to a more hydrophobic environment on complex formation. Excess
ABD-E346C-rACT was employed to avoid proteolysis of the complex by Chtr
(14). The estimated increase of fluorescence intensity at 502 nm on
full conversion of I to the E*I* complex is 46%. The emission spectrum
of ABD-S348C-rACT shows a somewhat larger increase (~2-fold) in
intensity and a small blue shift (~3 nm) on complexation with Chtr,
measured at pH 7.0 (data not shown). In contrast with complex
formation, HNE-cleavage of ABD-E346C-rACT produces a much smaller
increase in emission intensity (~13%) and a slight red shift (~3
nm) (Fig. 2).

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Fig. 2.
Fluorescence emission spectra.
i, intact ABD-E346C-rACT; ii, HNE-cleaved
ABD-E346C-rACT, prepared as described under "Experimental
Procedures"; iii, mixture of Chtr*·ABD-E346C-rACT*
(85%) and intact ABD-E346C-rACT (15%), formed by addition of ~0.9
equivalents of Chtr to 1.0 equivalent of ABD-E346C-rACT. Spectra were
taken in 20 mM NaPi, pH 7.5, with an excitation wavelength
of 384 nm. Inset, SDS-PAGE analysis of samples i-iii above
made up in reducing loading buffer (0.1% SDS, 5% glycerol, 44 mg/ml
ammediol, 2.5 mg/ml bromphenol blue, 7.25 mg/ml dithiothreitol). Bands
are as follows: C, Chtr*-ABD-E346C-rACT* complex;
I, intact ABD-E346C-rACT; Icl,
HNE-cleaved ABD-E346C-rACT.
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The fluorescence emission spectra shown in Fig. 2 were obtained at pH
7.5. Qualitatively similar results were obtained at pH values of 5.5 and 6.5.
Rates of Complex Formation with Chtr by Stopped-flow
Spectrofluorometry at pH 7, 25 °C and 40 °C--
Mixing of Chtr
and ABD-E346C-rACT at pH 7, 25 °C leads to a biphasic response, with
the fluorescence intensity at 480 nm first decreasing and then
increasing (Fig. 3). Rate constants for
both phases were calculated by fitting the data to Scheme II (Table II). Shown in Fig. 3 are results obtained
for initial concentrations of Chtr and ABD-E346C-rACT of 125 and 25 µM, respectively. Essentially the same results were
obtained when the Chtr and ABD-E346C-rACT concentrations were varied,
maintaining a 5:1 ratio, over a [Chtr] range of 25-125
µM (Table II) or at higher E:I ratios (data not shown),
demonstrating that both rate constants measure first-order processes,
i.e. processes taking place after formation of the encounter
complex (E·I) between ABD-E346C-rACT and Chtr. A similar biphasic
response was seen at 40 °C, with correspondingly higher values for
kI and kII.
As mentioned above, the SI for ABD-E346C-rACT interaction with
Chtr is slightly greater than 1. Formation of cleaved ABD-E346C-rACT was ignored in our calculations, a procedure that is justified not only
because the amount of such formation was small (~6%), as judged by
SDS-PAGE analysis (see below) but also because the fluorescence change
at 480 nm accompanying ABD-E346C-rACT cleavage (Fig. 2, sample
iii) was <15% that accompanying complex formation with Chtr.

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Fig. 3.
Kinetics of ABD-E346C-rACT-Chtr complex
formation at 25 °C, pH 7.0 (20 mM sodium
phosphate). Shown are normalized changes following rapid mixing of
Chtr (125 µM) and ABD-E346C-rACT (25 µM) in
(a) the fluorescence emission signal at 480 nm on excitation
at 384 nm (trace); (b) E*I* formation as measured
by SDS-PAGE ( ); (c) postcomplex fragment formation, as
measured by HPLC assay ( ); and (d) cleaved I formation as
measured by SDS-PAGE ( ). Solid lines represent best fits
to Scheme II of the fluorescence (black) and E*I* formation
(blue) data.
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Table II
Rate constants at pH 7
Fit to Scheme II or to single exponential. Error ranges shown are
average or precision (single determinations). Values for MCM-A352C-rACT
are from Ref. 26. Values for WT-rACT are from Ref. 11 and are measured
at pH 7.5. All other values are at pH 7.0.
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As contrasted with ABD-E346C-rACT, mixing ABD-S348C-rACT with Chtr
leads to a monophasic increase in fluorescence intensity at 480 nm that
is well fit to a single exponential (data not shown). In the
concentration range studied, using [Chtr] in excess over [ABD-S348C-rACT], the observed rate constant at 25 °C, 4.6 s
1, is independent of [Chtr] and corresponds to a first
order process (Table II). The similarity in this value to
kII above (4.2 s
1) leads us to
believe that these constants measure the rates of similar processes for
the two rACT derivatives. The failure of ABD-S348C-rACT to display a
fast first-order process on reaction with Chtr, corresponding to
kI above, may either be due to the absence of
such a process (i.e, direct conversion of E·I to E*I* in
Scheme II) or to its being undetectable by this approach
(e.g. if the
kI/kII ratio was greater
than or equal to that for ABD-E346C-rACT but little change in
fluorescence intensity accompanied E·I to EI
conversion). We believe the latter possibility more likely (see under
"Discussion"). Further kinetic studies, described below, were
conducted with ABD-346C-rACT, because this derivative offered the
greater promise for detecting intermediates on conversion of E·I to
E*I*.
Rate of ABD-E346C-rACT-Chymotrypsin Complex Formation by
Quenched-flow--
In an experiment exactly paralleling the
stopped-flow experiment described above, rapid quench kinetics was
employed to determine the rates of E*I* formation between Chtr and
ABD-E346C-rACT, by SDS-PAGE analysis (Fig.
4), and of postcomplex fragment
formation, by reverse phase-HPLC analysis. Both analyses gave virtually
identical results, consistent with our earlier demonstration for WT-ACT (2) that E*I* and postcomplex fragment are formed at the same rate.
Both data sets were fit to Scheme II (Fig. 3), using the Hopkinsim
program, yielding rate constants indistinguishable from those obtained
by fitting of the stopped-flow data (Table II). Thus, at pH 7 and
25 °C, stopped-flow and quenched stopped-flow kinetics are
consistent with formation of E*I* via Scheme II, in which fluorescence
intensities vary in the order E*I*> E·I> EI
and
neither the encounter complex nor the intermediate complex is
SDS-stable, thus accounting for the lag in SDS-stable complex
formation.

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Fig. 4.
Quenched-flow/SDS-PAGE analysis of
ABD-E346C-rACT reaction with Chtr at 25 °C, pH 7.0. The
concentrations were 25 µM for ABD-E346C-rACT and 125 µM for Chtr. C, Chtr*ABD-E346C-rACT* complex;
I, ABD-E346C-rACT; Icl,
cleaved ABD-E346C-rACT; E, Chtr. No bands migrating between
I and C were visible over the time period used for rate constant
calculation. Such bands are characteristic of E*I* complex proteolyzed
by Chtr (14). Numbers at bottom are times of quenching
(seconds). St, molecular mass standards. Lanes E
and I are Chtr and ABD-E346C-rACT, respectively.
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It was also possible to quantify the minor amount (~6%) of cleaved
ABD-E346C-rACT formed as a function of time by SDS-PAGE analysis (Fig.
3), although with less precision than for E*I* formation. Nevertheless,
it is clear that E*I* and cleaved I are formed at approximately the
same rate.
Rates of ABD-E346C-rACT-Chtr and WT-rACT-Chtr Complex Formation at
10 °C--
More extensive studies of rates of ABD-E346C-ACT
interaction with Chtr as a function of pH in the range 5-8 were
conducted at 10 °C to take advantage of slower rate constants that
might permit observation of more than one intermediate. Typical
stopped-flow spectrophotometric and quenched-flow data are displayed in
Fig. 5. Taken together, they provide
evidence for a pathway for E*I* formation (Scheme III) involving three
intermediates between the encounter complex E·I and the final E*I*
complex.
Considering first the results obtained at pH 6.5 (Fig.
5B), the observed fluorescence changes provide clear
evidence of a three-phase reaction, in which fluorescence first
increases rapidly on EIa formation, then slightly decreases
due to EIb formation, and finally increases again on
EIc formation. The clear lag in E*I* formation as compared
with the third phase of fluorescence change necessitates invocation of
a fourth phase of reaction
i.e. there are three
intermediates (EIa, EIb, and EIc)
between the encounter complex and E*I*
and implies that the
fluorescence of E*I* is similar to that of EIc. The same
pattern is seen at pH 7.0 (Fig. 5C), although here the lag
between the final phase of fluorescence change and E*I* formation is
much less pronounced. As the pH is raised toward 8.0 (Fig.
5D), the lag between the final phase of fluorescence change
and E*I* formation disappears, reflecting an increase in
k4 relative to k3.
Finally, as the pH is lowered toward 5.0 (Fig. 5A), the
phase corresponding to decreased fluorescence intensity disappears,
presumably because the fluorescence of EIb becomes greater
than or equal to that of EIa. On the other hand, the lag
between the final phase of fluorescence change and E*I* formation
becomes even more pronounced.

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Fig. 5.
Kinetics of ABD-E346C-rACT-Chtr complex
formation at 10 °C. Shown are normalized changes in
fluorescence emission at 480 nm on excitation at 384 nm
(traces) following rapid mixing of ABD-E346C-rACT (5 µM) and Chtr (50 µM). Also shown are
parallel SDS-PAGE measurements of E*I* formation ( ). At pH 5.0, the
latter measurements were also carried out for WT-rACT ( ).
A, at pH 5.0; B, at pH 6.5; C, at pH
7.0; D, at pH 8.0. Solid lines represent best
fits to Scheme III, as described under "Experimental Procedures."
In A, the bottom line is for ABD-E346C-rACT, and
the middle line is for WT-rACT. In D, the
bottom line is for E*I* formation, and the top
line is for fluorescence trace. Insets show the early
phase of the change in fluorescence emission. Buffers were as follows:
pH 6-8, 20 mM sodium phosphate; pH 5.0-5.5, 20 mM sodium acetate.
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Also shown in Fig. 5A are quenched-flow results for E*I*
formation from WT-rACT and Chtr at pH 5. As these data make clear, not
only is the lag phase seen with ABD-346C-rACT also seen with WT-rACT,
but the rates of E*I* formation in both cases are virtually identical.
At each pH, values for rate constants
k1-k3 for ABD-346C-rACT
reaction were obtained by fitting the stopped-flow fluorescence data to
the first three steps of Scheme III. Each of these constants is
first-order, describing processes following encounter complex formation, as shown by their insensitivity to changes in Chtr and/or
ABD-E346C-ACT concentration over the ranges employed (Table III). As above, the formation of cleaved
ABD-E346C-rACT was ignored. Values for k4 were
determined by fitting quenched-flow data to Scheme III, using values
for k1-k3 determined by
fitting the stopped-flow fluorescence data.
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Table III
Rate constants at 10 °C
Fit to Scheme III as described. Error ranges shown are average
deviations. Values in parentheses were fixed in calculating
k4.
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The observed pH dependences of rate constants
k1-k4 are displayed in
Fig. 6 and can be fit to Scheme IV,
allowing estimation of rate constants for interconversion of different
protonated forms of each of the five Chtr·ABD-E346C-rACT complexes
included in Scheme III. The bell-shaped curve for
k1 implies that conversion of the encounter
complex to the first intermediate complex proceeds most rapidly via a
neutral form, HE·I, and that either protonation of this complex to
form H2E·I or deprotonation to form E·I leads to a
large decrease in rate. Due to the narrowness of the pH optimum (i.e. the closeness of the two relevant
pKa values, 7.0 and 7.1), the maximum
fraction of the encounter complex in the most reactive form does not
exceed one-third, accounting for the observed maximal rate constant at
pH 7 being well below the calculated rate constant for HE·I.

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Fig. 6.
pH effects on first order rate
constants. A, k1; B,
k2; C, k3;
D, k4. All rate constants are from
Table III and are in s 1. Solid lines represent
best fits to Scheme IV.
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The rate constant for conversion of the first intermediate to the
second intermediate depends to only a minor extent on pH. Interpreted
according to Scheme IV, HEIa conversion to HEIb
proceeds about 1.5 times as fast as H2EIa
conversion to H2EIb, but deprotonation to
EIa has no effect on rate. Similarly, both EIb
and HEIb are converted to the third intermediate with the
same rate constant, but both of these reactions proceed much more
rapidly (10-fold) than the corresponding conversion of
H2EIb. Finally, conversion of the third
intermediate to E*I* proceeds most rapidly at high pH, with
EIc being >5-fold more reactive than HEIc,
which is in turn >3-fold more reactive than
H2EIc.
Fluorescence Intensities of Intermediates--
The fitting
procedure for the stopped-flow fluorescence data allows calculation of
relative fluorescence intensity differences at 480 nm for each
intermediate relative to E·I as a function of pH (Fig.
7). A large positive difference is seen
for EIc over the whole pH range, with the largest value
observed at pH 6.5. EIa shows a positive difference at pH
5, which falls to virtually nothing at pH 8. A more dramatic change,
however, is seen for EIb, in which a positive difference is
replaced by a negative difference as the pH is raised from 5 to 8. The
relative fluorescence intensities of both EIa and
EIb are modulated by a group (or groups) of apparent
pKa 6.5-7.0 (see under "Discussion").

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Fig. 7.
pH effects on normalized relative changes in
fluorescence intensities. EIa, ; EIb,
; EIc, . Values shown were obtained by subtracting
the fluorescence intensity of the encounter complex, E·I, from that
of the species indicated, and setting the value for the largest change
(EIc at pH 6.5) equal to 1.00.
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DISCUSSION |
Earlier, we showed that at pH 7.0 and 40 °C, the spectral
change resulting from MCM-A352C-rACT interaction with Chtr proceeded more rapidly than E*I* formation, allowing the inference that an
intermediate formed between the encounter complex E·I and the acyl
enzyme E*I* (26). The major result of the current study is the direct
demonstration that there are at least three identifiable intermediates,
EIa, EIb, and EIc, between the
encounter complex formed between ABD-E346C-rACT and Chtr and E*I*. It
might be argued that ABD-E346C-rACT interaction with Chtr reflects a
peculiarity due to the presence of a bulky group in the hinge region
and is not a good model for WT-rACT interaction. Strong evidence that this is not so comes from the similarities of the two molecules as Chtr
inhibitors, as judged by their ki and SI values (Table I) and their values for k3 and
k4 at pH 5 (Table III and Fig. 5A).
Furthermore, even though the complexity of Scheme III introduces
ambiguity into comparisons of overall first-order rate constants for
E*I* formation, it is worth noting the similar magnitudes of the single
rate constants observed for WT at pH 7.5 (7 ± 1 and 17 ± 2 s
1 at 25 and 40 °C, respectively) and
kII for ABD-E346C-rACT at pH 7.0 (4.6 ± 0.5 and 22 ± 2 s
1 at 25 and 40 °C, respectively)
(Table II). In addition, at pH 7 and 40 °C, values of
kI and kII for
ABD-E346C-rACT (53 ± 10 and 22 ± 2 s
1) are
similar to those estimated for MCM-A352C-rACT (30-45 and 20-25
s
1) (Table II), in which the bulky fluorescent group is
at P7, far from the hinge region.
Below, using the results of this study, especially Figs. 6 and 7, we
propose a model relating the structures of intermediates EIa, EIb, and EIc to conformational
changes within the RCL that are known to be crucial for the inhibitory
activity of a serpin and consider the question of from which of these
intermediates, if any, does partitioning occur between the inhibitor
and substrate pathways (Scheme I)?
A Proposed Model for Conformational Changes in the RCL on
Conversion of E.I to E*I* via EIa,
EIb, and EIc--
In formation of E*I* from E
and I we know, or can safely assume on the basis of prior work (7, 8),
that the following must occur: (a) the RCL assumes the
canonical conformation in fitting into the active site of Chtr;
(b) there is a major insertion of the RCL into
-sheet A
(s4A), although how much insertion occurs is unclear; (c)
the structure of Chtr within the complex changes considerably
vis-à-vis the native structure, with concomitant inactivation of
its catalytic apparatus (14-16); and (d) Ser-195 in Chtr is
acylated with ACT at residue P1 (11, 12), with formation of the
postcomplex fragment, a reaction that proceeds via a tetrahedral intermediate.
We propose that in Scheme III, E·I conversion to EIa
(step 1) principally involves rearrangement of the RCL to the required canonical conformation, which is followed by a conformational change
within Chtr to give EIb (step 2). In both EIa
and EIb, the P13 residue moves in and out of an inserted
position within sheet A, in a process modulated by the ionization state
of His-57. Formation of EIc (step 3) involves a closer
fitting of the RCL to the catalytic machinery of Chtr
(EIc), as well as insertion of a major portion of the RCL
within
-sheet A. Finally, conversion of EIc to E*I*
(step 4) involves covalent reaction between Chtr residue Ser-195 and
the P1 residue of ACT to give the tetrahedral intermediate and
ultimately acyl enzyme. Evidence for this proposal is discussed further below.
Relevance of Chtr Hydrolysis of a Simple Substrate to Steps 2-4 in
Scheme III--
Earlier, Fink (41) identified three intermediates
intervening between the encounter complex E·S and the acylated enzyme on reaction of
N-acetyl-L-Phe-p-nitroanilide and
Chtr at pH 7.6 in 65% aqueous dimethyl sulfoxide (Scheme
V).
At
56 °C, k2* > k3* > k4*
k5*, but on extrapolation to 25 °C, this
order changed to k2* ~ k3* ~ k4*
k5*. Moreover, over the pH range 4.2-7.8,
k2* and k3* were
insensitive to pH, whereas both k4* and
k5* increased with pH, with kinetically
determined pKa values of 5.9 and 7.6, respectively
(both measured at 0 °C). When Chtr was replaced with its N-Me His-57
derivative, which has very low enzymatic activity,
k2* and k3* were
unaffected, but reaction 4* could not be detected. Fink (41) concluded
that reactions 2* and 3* represented conformational changes within Chtr
following substrate binding, but that both reactions 4* and 5* involved
the Chtr catalytic apparatus. He assigned reaction 4* to the formation
of a pre-tetrahedral intermediate (In) that requires His-57 to be in
the basic form, and reaction 5* to the formation of acyl enzyme via a
tetrahedral intermediate that does not accumulate, arguing that In
could not itself correspond to a tetrahedral intermediate, because its
rate of breakdown increases as pH is increased. (In contrast,
tetrahedral intermediate does accumulate at high pH (9.4) during
acyl-elastase formation from elastase and p-nitroanilide
substrates (42).)
There is a striking degree of similarity in the properties of several
of the steps seen by Fink (41) and ourselves. The conversion of
EIa to EIb (step 2) in Scheme III parallels
steps 2* and 3* in Scheme V in taking place comparatively rapidly and in showing little pH dependence. Similarly, k3
and k4, the rate constants for EIb
conversion to EIc and EIc conversion to E*I*, respectively, depend on basic groups of apparent pKa values of 6.1 and
7.8 (Fig. 6), closely paralleling what Fink (41)
reports for k4* and k5*
in Scheme V. These similarities lead us to speculate that reactions 2, 3, and 4 in Scheme III parallel reactions 2* and 3*, 4*, and 5* in
Scheme V, respectively, such that the intermediates that accumulate
between E·I and E*I* do not involve covalent bond formation between
ACT and Chtr. Such a model agrees with conclusions reached by Stone and
Le Bonniec (43) for the reaction of the heparin complex of antithrombin with thrombin, but run counter to the model of Olson et al.
(44) for the reaction of plasminogen activator inhibitor-1 with
trypsin. This apparent disagreement may simply reflect real differences in the relative free energies of intermediates formed in the reactions of different proteinases, as noted above in connection with tetrahedral intermediate accumulation in Chtr versus elastase.
Step 1 in Scheme III--
In contrast to steps 2-4, step 1, the
formation of EIa, which is both rapid and has strong pH
dependence, has no clear analogue in Scheme V, allowing the inference
that step 1 represents a transformation unique to the interaction of
serpins with Chtr, the conversion of the RCL to the canonical
conformation. The necessity of such a conformational change is
suggested by the structure of an intact variant of rACT retaining
inhibitory activity, in which the RCL is present as a distorted
-helix (18). It would be reasonable for the rate of formation of the
canonical conformation to depend on the charge relay system being in
the catalytically active form (neutral His-57), accounting for the
ascending limb of the pH rate profile for k1
with apparent pKa 7.0 (Fig. 6A). Accounting for the descending limb (pKa 7.1) is less obvious. A requirement for the protonated form of one of the eight remaining His residues in the E·I complex (His-40 in Chtr and seven
His residues in rACT) is a possibility. Based on proximity within a
model of the docked rACT·Chtr complex (45) to either His-57 or
Ser-195 in Chtr, or to P13, the most likely candidates are His-40 in
Chtr and His-204, -224, or -225 in ACT.
The Magnitudes and Directions of Fluorescence Intensity Changes
(Fig. 7) Support the Proposed Conformational Changes--
According to
our model, motions within the active site would be coupled to the hinge
region of ACT on formation of EIa, so that the changes in
fluorescence intensity on conversion of E·I to E*I*, although
directly reflecting changes in the local environment of the ABD group
attached to Cys-346, could also detect changes occurring within the
active site of Chtr. We interpret increases and decreases in
fluorescence intensity in Fig. 7 as reflecting either further insertion
of the ABD group into
-sheet A or movement toward a more
solvent-exposed environment, respectively, based on the increase in
fluorescence we observe on E*I* formation (Fig. 2), which parallels
increases seen for fluorescent probes placed at residues P7 (26) and P9
(46). According to this interpretation, the fluorescence changes seen
in EIa and EIb as a function of pH reflect
small movements of the RCL that are modulated by the protonation state
of His-57, with the inactive, protonated form corresponding to more
insertion within the A-sheet and the neutral, active form corresponding
to less. The movement is more pronounced for EIb,
indicating a stronger coupling of the hinge region to the active site
following the presumed conformational change in Chtr in step 2. Full
exposure of the RCL when bound to the catalytically active form of Chtr
is consistent with the hypothesis that a fully exposed RCL is ideal for
binding residues P3-P3' in the canonical conformation required for
productive interaction with a serine proteinase active site
(47-49).
Facile movement of the hinge region of rACT in and out of
-sheet A
is also suggested by the contrast between the Wei et al. (18) structure of intact rACT, in which the hinge region is fully
exposed, and the difficulty we find in derivatizing T345C-rACT, an
indication that, in solution, the P14 residue is at least partially inserted within
-sheet A. In addition, structural and fluorescence spectroscopy studies show that the binding of heparin to antithrombin induces a conformational change that results in the expulsion of an
inserted P14 residue into a solvent-exposed position (48, 50), and
model building studies of several intact serpin-proteinase complexes
show that it is possible to insert the RCL only as far as P12 while
maintaining residues P3-P3' in the canonical conformation (40). This
latter result offers a rationale for the failure of ABD-S348C-rACT, the
P11 derivative, to show a change in fluorescence on a time scale
appropriate for EIb formation on interaction with Chtr at
pH 7, because an ABD group at P11 would be expected to remain exposed
on conversion of I to EIb.
In contrast to the pH-dependent changes seen with
EIa and EIb, the ABD-group in EIc
shows a large increase in fluorescence intensity versus
E·I over the entire pH range investigated. The magnitude of this
change, its relative insensitivity to pH, and the fact that no further
fluorescence change is detectable on conversion of EIc to
E*I* suggest that it reflects the major insertion of the RCL within
-sheet A that accompanies overall E*I* formation. Thus, contrary to
an earlier suggestion (29), RCL insertion appears not to be directly
coupled to the inactivation of the Chtr catalytic apparatus, resulting
in E*I* formation, because such inactivation would be expected to block
the conversion of EIc to tetrahedral intermediate and acyl enzyme.
From Which Intermediate Does Partitioning Occur?--
Earlier (11)
we presented evidence that in the interaction of rACT with Chtr,
partitioning between the inhibitor and substrate pathways, leading to
formation of the inhibited complex E*I* or the cleaved inhibitor
Is, respectively, proceeds from a common intermediate
formed in or following a step that is largely rate-determining. The
current result showing that the rate of formation of the minor product, cleaved ABD-E346C-rACT, is essentially the same as the rate of formation of the major product, E*I* (Fig. 3) supports this notion. As
step 3 in Scheme III is largely or completely rate-determining for E*I*
formation over the whole pH range investigated (Table III), the
possible candidates for the partitioning intermediate are
EIc and an as yet unobserved intermediate that occurs
between EIc and E*I*: e.g. the tetrahedral
intermediate, or a putative "active" form of acyl enzyme that
either isomerizes to E*I* or hydrolyzes to E and I*. Although we
consider the latter possibility most likely, because it preserves the
catalytic apparatus necessary for the relatively rapid covalent
reactions in E*I* formation (which occur at essentially the same rate
as in substrate turnover; see Ref. 11), a clear challenge for future
work will be to convincingly distinguish among these possibilities.