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INTRODUCTION |
1-Antichymotrypsin
(ACT)1 is a member of the
serine proteinase inhibitor, or serpin, family that typically forms
very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*)
with its target proteinases. Serpins share a conserved tertiary
structure in which an exposed region of amino acid residues (called the
reactive center loop or RCL) acts as bait for a target proteinase. For ACT, this loop extends from residues 342 to 367, denoted P17-P9', in
which, following the nomenclature of Schechter and Berger (1), the scissile bond cleaved by the targeted proteinase chymotrypsin (Chtr) is between the P1 and P1' residues. The native serpin structure is unusual in that it is metastable and is considered to be in a
stressed (S) conformation. The cleaved serpin released from the complex is much more stable than the intact serpin (2, 3) and is
considered to be in a relaxed (R) conformation. Noteworthy among many structural differences between the S and
R conformations (4) is the A
sheet, which is converted
from a five-strand sheet into an antiparallel six-strand sheet by the
insertion of RCL residues P1-P14 as strand s4A. As a result of this
insertion, the P1 and P1' residues are separated by 70 Å.
There is now very good evidence that within E*I* the two proteins are
linked covalently; an acyl enzyme has formed following attack by the
nucleophilic Ser195 of the serine proteinase on the P1
residue of the serpin (5-7). This species is formed rapidly (5) and is
formally similar to the acyl enzyme species normally seen as an
intermediate in serine proteinase catalysis. However, its subsequent
hydrolysis is extremely slow, resulting in the observed inhibitory
effect of serpins. This "trapped" acyl enzyme results from
structural changes within the enzyme leading to distortion of the
active site, which was inferred from proteolysis (8, 9) and NMR (10)
studies in solution and recently confirmed by a crystal structure of
the trypsin*antitrypsin* complex (11). Within this structure,
the antitrypsin is in the R conformation, and the trypsin attached to the P1 residue has translocated from the top of the serpin
molecule, defined as the position of the RCL in the intact, active
serpin, across its entire length to the bottom of the molecule.
The trypsin*antitrypsin* structure provides strong support for
one side in what has been an ongoing debate concerning the extent of
RCL insertion within the E*I* complex, whether full insertion
(R conformation), as first proposed by Wright and Scarsdale (12), or partial insertion, as suggested by Whisstock et al. (13). Earlier experimental results supporting each point of view are
summarized in Stone et al. (14). More recently,
studies employing fluorescence resonance energy transfer (FRET) (15) and donor-donor energy migration (16) have given results supporting the
full insertion model, whereas monoclonal antibody binding studies to
defined serpin epitopes (17, 18) favor the partial insertion
model. These continuing disagreements raise questions as to whether the
structure of the E*I* complex may be different for different serpins
and to what extent the time scale employed in measurement affects the
structure observed, i.e. whether the partially inserted form
may be an intermediate on the way to full insertion (19).
Here, we report time-resolved stopped-flow FRET and rapid mixing/quench
studies of E*I* formation between 
ACT, derivatized with a
fluorescence donor at the P1' position, and 
Chtr, derivatized with
a fluorescence acceptor at the amino terminus. Our results demonstrate
multistep formation of the E*I* complex in which the distance
separating the two fluorophores is inconsistent with formation of the
fully inserted R conformation. In consequence, we propose a
mechanism for E*I* formation that rationalizes the apparent
disagreements mentioned above. The chief features of this mechanism are
that: (a) partial insertion of the RCL occurs with the
P1-P1' peptide linkage intact; (b) acyl enzyme formation occurs following partial insertion and is not concomitant with full
insertion; and (c) the R conformation forms
reversibly, but not necessarily rapidly, from the partially inserted conformation.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine
N-p-tosyl-L-leucine chloromethyl
ketone-treated
-chymotrypsin,
-chymotrypsinogen, all chromophoric
proteinase substrates, N-
-tosyl-L-lysine
chloromethyl ketone (TLCK), dithiothreitol, quinine sulfate,
hydroxylamine hydrochloride, and phenylmethylsulfonyl fluoride were
obtained from Sigma. Bovine
N-p-tosyl-L-phenylalaninechloromethyl ketone-treated trypsin was obtained from Worthington. The
concentrations of these enzymes and of rACT were determined as
described earlier (20). All HPLC solvents,
H2O2, and formic acid were obtained from Fisher
Scientific. 4-Bromomethyl-7-methoxycoumarin (BMMC) and
7-diethylaminocoumarin-3-carboxylic acid (DEACA)-succinimidyl ester
were acquired from Molecular Probes, Inc. (Eugene, OR). SDS-PAGE
analysis was performed according to Laemmli (21). Standard proteins
were from Bio-Rad.
Construction, Expression, and Purification of
rACTs--
S359C-rACT was constructed using sequence overlap
expression polymerase chain reaction and the ACT expression vector
described previously (20, 22). The internal primers coding for
the Ser
Cys mutation are as follows:
5'-CCCTCCTTTGTGCATTAGTGGAGACA-3' and
5'-GCACAAAGGAGGGTGATTTTGAC-3' (the mutation sites are in boldface). The polymerase chain reaction product, representing the
entire coding region, was cut with BstXI, gel-purified, and inserted in the correct reading orientation in pZMS. Full gene sequencing confirmed a single codon change. S359C-rACT and rACT were
purified to homogeneity as described earlier (22).
Preparation of Derivatized S359C-rACT--
S359C-rACT (2 µM in 50 mM Tris, 50 mM KCl, pH
8.3) was reacted with a 100-fold excess of BMMC for 12 h at
0 °C in an amber-colored reaction vessel. Samples were concentrated
and buffer-exchanged using Amicon, Centriprep-30, and Centricon-30
concentrators (Amicon, Inc.). The stoichiometry of
(7-methoxycoumarin)-methyl (MCM) labeling was determined by absorbance
measurements. Total bound MCM concentration was determined using
330 = 13,000 M
1
cm
1. Total protein concentration was
determined using
280 = 39,000 M
1 cm
1,
calculated as the sum of the coefficients of the unlabeled protein (36,000 M
1
cm
1, calculated from an ACT solution
standardized by Bradford analysis) and the MCM group, determined using
the A280/A330 ratio for
BMMC. Purified protein had a calculated stoichiometry of 1.05 MCM/protein. As shown earlier (22, 23), the unique Cys residue in
wild-type rACT, Cys237, is buried in the hydrophobic core
of the molecule and has negligible reactivity under the reaction
conditions used for derivatization.
Preparation of Derivatized
-Chymotrypsin--
Zymogen was
derivatized prior to activation, permitting specific labeling of the
unique amino terminus present in the proenzyme. Chymotrypsinogen (19 µM in 20 mM sodium Pi, pH
7) was reacted with a 6-fold excess of
7-diethylaminocoumarin-3-carboxylic acid, DEACA-succinimidyl ester at
27 °C for 30 to 150 min in a foil-covered reaction vessel. The
reaction was quenched by adding a 0.1 volume of 1.5 M
hydroxylamine, pH 8.5, with stirring at room temperature for 1 h.
The sample was then washed and concentrated using Amicon and
Centricon-10 concentrators. Total bound DEACA concentration was
determined using
432 = 44,000 M
1 cm
1
(24). The
280 of labeled protein, 58,000 M
1 cm
1,
was calculated as the sum of the extinction coefficients of the
unlabeled protein (50,000 M
1
cm
1) (25) and the DEACA group (8,000 M
1 cm
1,
as determined by the
A280/A432 ratio for
DEACA). The proenzyme had a calculated stoichiometry of 0.50-1.2
DEACA/protein depending on the time of incubation employed. All of the
kinetics experiments reported in this paper were conducted with 0.75 DEACA/Chtr, prepared using a 30-min incubation time. Labeled proenzyme
(8 µM) was activated by incubation with trypsin (0.4 µM) at 27 °C in 100 mM Tris, pH 7.6, until
maximum activity was achieved (~90 min). The sample was then quenched
with TLCK (final concentration, 2 mM), concentrated, and
buffer-exchanged using Amicon, Centriprep-10, and Centricon-10 concentrators (Amicon, Inc.).
Chtr Enzymatic Activity--
Kinetic parameters
Km and kcat were evaluated by
Eadie-Hofstee plots for
-CT and DEACA-
-CT using the chromophoric substrate succinimidyl-AAPF-p-nitroanilide in 20 mM sodium phosphate, pH 7.0. The stoichiometry of active
site Ser195 in DEACA-
-Chtr was determined by titration
with
4-methylumbelliferyl-p-(N,N,N-trimethylammonium)-cinnamate (26). All assays were performed at room temperature (20 ± 1 °C).
Characterization of Derivatized S359C-rACT--
Second-order
rate constants for inhibition ki and stoichiometry
of inhibition (SI) were determined for all serpin-proteinase pairs.
Inhibition rate constants were determined by incubating equimolar
concentrations of enzyme and inhibitor under second-order conditions
and removing aliquots for residual enzyme activity determination as
described earlier (20). SI values were determined by densitometric
analysis of SDS-PAGE gels by comparing the intensity of cleaved serpin
following complex formation with proteinase with the band intensity of
unreacted intact serpin (23).
Mass Spectral Analysis--
Electrospray ionization was
performed on a Micromass Platform LC Electrospray mass spectrometer
(Micromass® UK, division of Waters Corp., Milford, MA) at
the Mass Spectrometry Facility of the Department of Chemistry at the
University of Pennsylvania. MALDI-TOF (matrix-assisted laser
desorption ionization time-of-flight mass spectrometry) was performed
on a VG Tofspec (Fisons Instruments, Danvers, MA) at the Protein
Chemistry Laboratory in the Medical School of the University of Pennsylvania.
Oxidative Liberation of the Amino-terminal Fragment
Cys1-Leu13 from
-Chtr--
Oxidation of
chymotrypsin with performic acid results in cleavage of the disulfide
links in the protein and allows for the separation of the
amino-terminal fragment (
-chain) from the rest of the molecule (27,
28).
-Chtr or DEACA-
-Chtr was suspended in formic acid (88% in
H20), and a 2-fold volume of performic acid (prepared by
adding 1/20 volume 30% H202 to formic acid
followed by room temperature incubation for 1 h (29, 30)) was
added, bringing the final concentration of enzyme to 20 mg/ml.
Following incubation on ice for 15 min, the reaction mixture was
brought to pH 5 by the addition of 10 volumes of 1 M Tris,
pH 10, leading to virtually complete precipitation of residues
Ile16-Asn245 (27, 28), as shown by subsequent
HPLC analysis of the supernatant (see below). The precipitate was
removed by centrifugation and redissolved in formic acid, and the
amount of incorporated probe was determined by
A430 measurement. The supernatant was subject to
RP-HPLC (Rainin C-18 column, Microsorb-MVTM, 50 × 4.6 mm, 300 Å, 5 µm). Following equilibration with 0.1% trifluoroacetic
acid in water, peptides were eluted with the following gradient of
acetonitrile (also containing 0.1% trifluoroacetic acid): 0-10 min,
0-20%; 10-30 min, 20-30%; 30-90 min, 30-40%; 90-95 min,
40-100%; 100-105 min, 100%. HPLC data were analyzed using
Turbochrom Navigator software from PerkinElmer Life Sciences. The
amounts of
-chain were estimated from peak areas at 215 nm, corrected for elution yields of both the modified (23.4 ± 1.8%) and unmodified (71.1 ± 5.8%) forms, taking into account the
contributions to A215 from peptide bonds, side
chains (31), and fluorescent probe.
Stopped-flow Fluorescence--
Stopped-flow fluorescence
emission traces were acquired using an Applied Photophysics
SX.18MV stopped-flow spectrofluorometer with excitation at 330 and 430 nm. Emission was monitored at 400 nm (the emission maximum of
MCM-S359C-rACT) or 475 nm (the emission maximum of
DEACA-
-chymotrypsin). Traces were fit to a three-step kinetic model
using Pro-Kineticist software (Applied Photophysics Ltd.).
Steady-state Fluorescence Spectra--
Serpin-proteinase
complexes were formed at 21 °C in 20 mM sodium
Pi, 10 mM EDTA, pH 7, by the addition of 5 µM proteinase (50 µl) to 20 µM serpin (50 µl) and incubation for 15 min. Static fluorescence spectra were
acquired using a PerkinElmer LS50 luminescence spectrometer with
excitation at 330 nm.
Rapid Mixing Quenched Flow--
Rapid quenched-flow kinetic
studies were carried out using a KinTek Chemical-Quench-Flow Model
RQF-3 machine followed by densitometric analysis on SDS-PAGE as
described previously (22). SDS-PAGE analysis was performed on aliquots
of the acid-quenched reaction mixture using gels containing 12%
polyacrylamide. Prior to analysis, all samples were precipitated with
freshly prepared 20% trichloroacetic acid, incubated on ice 30 min, centrifuged and redissolved in 10 µl of 20 mM sodium
Pi, pH 7, to which 2 µl of 1 M Tris base was
added to neutralize the excess trichloroacetic acid present in the
quenched samples. Phenylmethylsulfonyl fluoride at a final concentration of 2 mM was also added to rapidly inactivate
any renatured chymotrypsin. Gels were stained overnight in GelCode® Blue Stain Reagent (Pierce), destained overnight in water, then dried prior to densitometric analysis. Data were fit to a three-step kinetic scheme using Hopkinsim, version 1.7 (32).
Measurement of FRET Efficiency--
Energy transfer was
determined by the acceptor enhancement method as well as by the
decrease in donor fluorescence (33). In calculating efficiencies,
observed values of fluorescence were corrected for the inner filter
effect (Equation 1) (33), in which
AEX is the absorption of all species at
D1 and AEM is the absorption of
all species at the emission wavelength (
D2). Such
corrections varied from 3 to 19% for both acceptor and donor fluorescence (see below).
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(Eq. 1)
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Fluorescence acceptor efficiencies (34) were calculated by
Equation 2, in which FDA is the fluorescence of
the donor/acceptor pair (MCM-S359C-rACT/DEACA-chymotrypsin) when
irradiated at the excitation wavelength of the donor and detected at
the emission maximum of the acceptor
(
refers to
330 nm excitation, 475 nm emission); FA is the
fluorescence of acceptor in the absence of donor: i.e.
unlabeled rACT/DEACA-chymotrypsin, using the same excitation/emission
wavelength pair; AD(
D1) and
AA(
D1) are the absorbances of
the donor and acceptor, respectively, at an excitation wavelength
(
D1) of 330 nm; and fa (equal
to 0.75) is the fraction of labeled acceptor protein.
|
(Eq. 2)
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The decrease in donor efficiency (35) was calculated by Equation 3, in which FDA is the fluorescence of the donor/acceptor pair, with irradiation at the excitation wavelength of the donor and
detection at the emission maximum of the donor
(
= 330 nm
excitation, 400 nm emission) and FD is the
fluorescence of the donor in the absence of acceptor: i.e.
MCM-S359C-rACT/unlabeled chymotrypsin, using the same
excitation/emission pair.
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(Eq. 3)
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Calculation of Distance--
The measured efficiency is a
function of the distance between donor and acceptor, R,
equal to [E
1
1]1/6
ro, where RO, the Förster distance
at which the efficiency is 50%, is calculated from Equation 4.
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(Eq. 4)
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In this equation, J, the overlap integral that
relates the degree of spectral overlap between the emission spectrum of
the MCM group covalently bound to serpin and the absorption spectrum of
the DEACA group attached to proteinase, was determined to be 4.52 × 10
14
M
1
cm
1nm4 using the method of Conrad
and Brand (36);
D, the quantum yield of the donor
in the absence of acceptor, was determined to be 0.023 for labeled
serpin complexed to unlabeled proteinase by comparison with quinine
sulfate as standard (37); n, the index of refraction of the
solvent, is assumed to be 1.33 (38); and
2 is the
orientation factor, describing the relative orientation in space of the
transition dipoles of the donor and acceptor.
2
may be set equal to 0.667 for donors and acceptors that randomize by
rotational diffusion prior to energy transfer or to 0.476 assuming that
a range of static donor-acceptor orientations exists that does not
change during the lifetime of the excited state (33). Using either of
these values gives similar values for RO of 23.5 Å (
2 = 2/3) or 21.0Å (
2 = 0.476).
Modeling--
Modeling of the MCM-S359C-rACT·DEACA-
-Chtr
complex was performed using the program Quanta version 98.1111 (Molecular Simulations, Inc., San Diego, CA). The coordinates for the
ACT-Chtr docked model proposed by Katz and Christianson (39) were used
as a starting point. The fluorescent probes were constructed in the two-dimensional Sketcher application and then converted to molecular structure files. These were simultaneously imported into Quanta along
with the published model, moved into proximity with their attachment
sites, and covalently attached with the modeling palette. The Molecular
Editor tool was used to add or remove hydrogens as necessary for proper
valency. The initial orientation of the probes was varied for both
probes by a rotation around the bond directly connected to the fluor in
90o increments, generating a total of 16 structures. The
probe aromatic rings were constrained to remain planar during the
minimization and subsequent energy calculations. Each structure was
then minimized first by the method of Steepest Descents, followed by
the method of Adopted-Basis Newton-Raphson. These structures were then
used to measure an average center-to-center distance between the probes in the encounter complex.
 |
RESULTS |
Site of DEACA Labeling of
-Chtr--
The site or sites of
labeling within
-Chtr depended on the overall stoichiometry of
chymotrypsinogen labeling. Preparations having
1.0 DEACA groups per
Chtr molecule had measurable labeling of the
Ile16-Asn245 polypeptide chain, rising to
~0.22 mol/mol for the sample containing 1.2 DEACA groups per Chtr.
This most likely reflects partial labeling of some of the 14 free Lys
residues present. On the other hand, preparations containing
0.75
DEACA groups per chymotrypsinogen molecule showed no (<0.01 mol/mol)
such labeling of Ile16-Asn245. For these
samples, all labeling was confined to the
-chain (residues 1-13)
and, by inference, to the only amino group within this chain, the
-amino group of Cys1. Accordingly, preparations
containing 0.75 DEACA per
-Chtr were used in all kinetics experiments.
Direct determination of the stoichiometry of
-chain labeling was
provided by RP-HPLC analysis of the soluble fraction of a performic
acid oxidized sample of DEACA-
-Chtr (Fig.
1) as described under "Experimental
Procedures." An analysis of the 75% labeled DEACA-
-Chtr gave a
labeled
-peptide/unlabeled
-peptide ratio of 3:1, measured by
corrected peak area at A215. The
assignment of peak 2 as the DEACA-labeled
-chain was indicated by
its absorbance at 430 nm and confirmed by mass spectral analysis (1589, M + 2 Na). The assignment of peak 1 as an underivatized
-chain was also confirmed by mass spectral analysis (1301, M + H).

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Fig. 1.
RP-HPLC separation of DEACA-modified
N-terminal fragment (Cys1-Leu13) from unmodified
fragment. Overlay of traces monitored at
A215 (a) and
A430 (b). Following oxidation of
DEACA- -Chtr (0.8 µM) with performic acid as described
under "Experimental Procedures," the supernatant was
analyzed by RP-HPLC. The sequence of elution was: 0-10 min, 20%
A; 10-30 min, 30% A; 30-90 min, 40%
A; 90-95 min, 100% A. A is 0.1%
trifluoroacetic acid in water, and B is 0.1%
trifluoroacetic acid in acetonitrile. The amounts of -chain were
estimated from peak areas at 215 nm, corrected for elution yields of
both the modified (23.4 ± 1.8%) and unmodified (71.1 ± 5.8%) forms.
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Functionality of Fluorescently Labeled Proteins--
As shown by
the results in Table I,
MCM-S359C-rACT retains full activity toward Chtr inhibition, as
measured by both the second-order rate constant for inhibition,
ki, and the SI of ~1. Similarly, DEACA-
-Chtr
retains full activity toward rACT and toward hydrolysis of the standard
substrate
N-succinimidyl-AAPF-p-nitroanilide (data
not shown). Finally, the reaction of MCM-S359C-rACT with DEACA-
-Chtr
has an SI value (~1) and a second-order rate constant for inhibition
(Table I) that are quite similar to those found for rACT reaction with
-Chtr.
Fluorescence Spectra of DEACA-
-Chtr, MCM-S359C-rACT, and the
DEACA-
-Chtr*MCM-S359C-rACT* Complex--
Relative to the sum
of the contributions of single-labeled complexes
DEACA-
-Chtr*wt-rACT* and
-Chtr*MCM-S359C-rACT*, excitation at 330 nm of the DEACA-
-Chtr*MCM-S359C-rACT* complex formed at 25 °C gives a decreased fluorescence at 400 nm, the donor emission maximum, and a comparable increased fluorescence at 475 nm, the acceptor emission maximum (Fig. 2). These
changes provide clear evidence for FRET within the complex, with
efficiencies, calculated as described under "Experimental
Procedures," of 0.30 for donor and 0.33 for acceptor. Similar
efficiencies of 0.32 and 0.36 for donor and acceptor were obtained for
the DEACA-
-Chtr*MCM-S359C-rACT* complex formed at 40 °C.
Incubations were performed in the presence of excess inhibitor to
prevent proteolysis of the E*I* complex (9).

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Fig. 2.
Fluorescence Spectra.
a, DEACA- -Chtr*MCM-S359C-rACT*; b,
-Chtr*MCM-S359C-rACT*; c, DEACA- -Chtr*rACT*;
d, sum of single-label complexes. Complexes were formed by
the addition of 5 µM proteinase to 20 µM
serpin in 20 mM sodium Pi, 10 mM
EDTA, pH 7.0, at 25 °C. Spectra were taken after 15-min incubations
and were corrected for internal filtering as described under
"Experimental Procedures" using Equation 1. Excitation was at 330 nm.
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Rates of Serpin·Proteinase Complex Formation by Stopped-flow
Fluorescence--
On donor excitation at 330 nm, the fluorescence
changes that follow mixing of MCM-S359C-rACT with
DEACA-
chymotrypsin at pH 7, 5 °C are triphasic between 2-5000
ms, whether monitored at 400 nm (Fig.
3A, donor fluorescence) or at
475 nm (Fig. 4A, acceptor
fluorescence). In the former case, intensity first decreases and then
increases in two steps, whereas in the latter case, fluorescence intensity increases in all three steps. Three phases are also clearly
evident on mixing MCM-S359C-rACT with
-Chtr (Fig. 3A, excitation at 330 nm, emission monitored at 400 nm). Shown in Fig.
3B is the calculated decrease in donor fluorescence due to FRET.

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Fig. 3.
Kinetics of complex formation by donor
fluorescence at 400 nm. A, lower trace, relative
fluorescence on adding MCM-S359C-rACT to DEACA- -Chtr. Upper
trace, relative fluorescence on adding MCM-S359C-rACT to -Chtr.
B, calculated decrease due to FRET, by subtracting
upper trace in A from lower trace in
A. Fluorescence at full reaction of MCM-S359C-rACT with
-Chtr is set equal to 1.0. Traces in A were corrected for
internal filtering as described under "Experimental Procedures"
using Equation 1. For panel A, the inset shows
expanded time scale, and solid lines are best fits to Scheme
1, with rate constants k1 = 27.5 s 1, k2 = 2.5 s 1, k3 = 0.6 s 1. Data shown were obtained at 5 °C, pH
7.0, on a combination of 10 µM inhibitor with 20 µM enzyme. Excitation was at 330 nm.
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Fig. 4.
Kinetics of complex formation by acceptor
fluorescence (475 nm) and E*I* formation. A, normalized
fluorescence change (uncorrected) on adding MCM-S359C-rACT to
DEACA- -Chtr with excitation at 330 nm (solid trace);
solid circles represent the fractional conversion to
MCM-S359C-rACT*DEACA- -Chtr*, following the addition of
MCM-S359C-rACT to DEACA- -Chtr, as determined by quenched stopped
flow, using SDS-PAGE and densitometric analysis (10). B,
typical SDS-PAGE results. Lane 1, molecular mass
standards; lanes 2-4 show incubation of MCM-S359C-rACT and
DEACA- -Chtr for 0.1, 1.25, and 5 s, respectively, prior to acid
quenching. Following quenching, samples were treated with
phenylmethylsulfonyl fluoride and precipitated with trichloroacetic
acid. The appearance of a doublet at 45 kDa (white arrow)
was an artifact of trichloroacetic acid precipitation. Data shown were
obtained at 5 °C, pH 7.0, on a combination of 10 µM
inhibitor with 50 µM enzyme.
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Rate constants for all three phases were calculated by fitting the
observed fluorescence data to Scheme 1, in which the encounter complex,
E·I, is separated from the final complex, E*I*, by two intermediates,
EIa and EIb (Table
II). The evident saturation in
rate constant values as the [E]/[I] ratio is increased from 2 to 5 provides clear evidence for three first order processes following E·I
formation.
Identical stopped-flow fluorescence experiments were also carried
out with acceptor excitation at 430 nm and monitoring at 475 nm
following addition of either MCM-S359C-rACT or rACT to DEACA-
Chtr.
In both cases, observed fluorescence increased, but the changes were
much smaller than those seen in Figs. 3 and 4, as would be expected for
a group at the amino terminus of a protein that is probably not
directly involved in protein-protein contacts. As a result, although
the data obtained could also be fit to Scheme 1 (Table II), the rate
constant values obtained for k3 were
considerably less precise.
Rate of DEACA-
-Chtr*MCM-S359C-rACT* (E*I*) Formation by
Rapid Mixing Quenched Flow--
In experiments exactly paralleling the
stopped-flow experiments described above, rapid quench kinetics was
employed to determine the rates of E*I* formation between
DEACA-
Chtr and MCM-S359C-rACT using SDS-PAGE analysis of aliquots.
The results (Fig. 4A), displayed alongside the acceptor
fluorescence results, show that E*I* formation occurs only after most
of the change in fluorescence has already taken place. Data were fit to
Scheme 1, assuming that only E*I* is SDS-stable, accounting for the
observed lag phase. Values of k3 were obtained
using values for k1 and
k2 derived from stopped-flow analysis. SDS-PAGE
analysis also showed no evidence for proteolysis of the E*I* complex
(9) over the 5-s period required for full E*I* formation (Fig.
4B) despite proteinase being present in excess.
Calculation of Energy Transfer Efficiency and
Distance--
Apparent FRET efficiencies as a function of time,
calculated from Equations 2 (acceptor) or 3 (donor) and stopped-flow
traces as described under "Experimental Procedures" were fit to
Scheme 1 using the rate constant values determined above, by direct
fitting of observed fluorescence and quenched-flow results (Fig.
5), to yield efficiencies for E·I,
EIa, EIb, and E*I* (Table
III) as well as estimates of the
distances between donors and acceptors in these species. The results
show that efficiencies increase monotonically in the conversion of
E·I to EIa, to EIb, and finally to E*I*, with
correspondingly small decreases in the estimated distances separating
the fluorophores. Also noteworthy is that the efficiency for E*I* of
0.33, measured from both donor and acceptor fluorescence at 5 °C
(Table III), is virtually identical to the values of 0.30-0.32 and
0.33-0.36 determined above (see Fig. 2) at 25 and 40 °C,
respectively. This identity is strong evidence that incubation at 25 and 40 °C for 15 min does not result in any major conformational
change detectable by FRET in the structure of the E*I* complex formed within 5 s at 5 °C (Fig. 5).

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Fig. 5.
FRET efficiency profile. Time-resolved
efficiency was calculated from stopped-flow traces corrected for
internal filtering and 75% labeling of DEACA- -Chtr. A,
changes observed at 475 nm upon the addition of MCM-S359C-rACT to
DEACA- -Chtr with excitation at 330 nm, efficiency calculated using
Equation 2. B, changes observed at 400 nm upon the addition
of MCM-S359C-rACT to DEACA- -Chtr with excitation at 330 nm, with
efficiency calculated using Equation 3. Data were obtained at 5 °C,
pH 7.0, on the reaction of 10 µM inhibitor with 20 µM enzyme. Solid lines are best fits to Scheme
1, with rate constants k1 = 27.5 s 1, k2 = 2.5 s 1, k3 = 0.6 s 1.
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Structure Modeling--
Although no crystal structure is available
for the ACT·Chtr complex, the docked model for this complex proposed
by Katz and Christianson (39) allows an estimation of the expected
chromophore-to-chromophore distance on noncovalent complex formation,
corresponding to one of the complexes, E·I, Eia, or
Eib, shown in Scheme 1. In the docked model, the distance
between the ACT Ser359 side chain oxygen and the
amino-terminal
-amino group in
-Chtr is 33.6 Å. In our modeling,
Ser359 was first mutated to Cys359, an MCM
group was attached to ACT-Cys359, and a DEACA group was
attached to the amino-terminal
-amino group in Chtr. Energy
minimization, performed using a large variety of starting orientations,
gave an estimated MCM center-to-DEACA center distance of 35.8 ± 2.0Å, in reasonable accord with the range of values estimated by FRET
for the E·I, Eia, or EIb complexes (26.4-34.6 Å, Table III).
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DISCUSSION |
Kinetic Scheme--
Recent pH-dependent studies of the
reaction of a fluorescent derivative of a cysteine variant of rACT at
the P13 position with Chtr enabled us to formulate a scheme for rACT
interaction with Chtr (Scheme 2), similar to Scheme 1 but containing
three intermediates between E·I and E*I* (23). The nature of the pH dependence of the fluorescent changes and the similarity of Scheme 2 to
the interaction of substrates with Chtr (40, 41) led us to propose that
E·I conversion to EIa principally involves rearrangement
of the RCL to the required canonical conformation. This
rearrangement is followed by two conformational changes, corresponding
to EIb and EIc formation, that result in a
closer fitting of the RCL to the catalytic machinery of Chtr, as well as to insertion of a major portion of the RCL within the A
-sheet (EIc); and also followed by conversion of EIc
to E*I*, corresponding to covalent reaction between Chtr residue
Ser195 and the P1 residue of ACT to give acyl enzyme.
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Our current results are consistent with Scheme 1. In Luo et
al. (23), the time dependence of fluorescent change and of E*I*
formation, measured at pH 7 and 10 °C, could be adequately fit with
three rate constants, and it was only in considering all of the rate
data collected over the pH range of 5.0 to 8.0 that the need for four
rate constants became clear. Thus, it is appropriate to compare rate
constants k1 (27.5 s
1), k2 (2.5 s
1), and k3 (0.6 s
1) measured in this work with rate constants
k1 (75 s
1),
k2 (5.6 s
1), and
k3, app (0.7 s
1),
respectively, measured in Luo et al., where
k3, app, the overall rate constant for
conversion of EIb to E*I*, is equal to
k3k4/(k3 + k4). This comparison shows each of the
constants measured at 5 °C in the present work to be lower within a
factor of 2 ± 1 than the values measured by Luo et al.
at 10 °C, a reasonable result for a conserved kinetic scheme.
A General Mechanism for E*I* Formation--
The results collected
in Table III indicate a small decrease in distance between the
fluorophores attached to the amino terminus of Chtr and P1' on
conversion of the E·I encounter complex to the SDS-stable E*I*
complex. This is consistent with only small-scale movement of Chtr
relative to ACT during this conversion, but it would not exclude larger
movements that would leave the fluorophore-fluorophore distance little
changed, such as those that could accompany partial insertion of the
RCL in the A
-sheet. What is excludable is a large-scale movement of
the enzyme across the length of the serpin molecule, as in the fully
inserted (R conformation) model proposed by Wright and
Scarsdale (12) and seen recently in the high resolution crystal
structure of the trypsin*antitrypsin* complex (11). This
conclusion is independent of the value of
2 in Equation 4. Above (see "Experimental Procedures"), we have provided the
rationale for using
2 values of 0.667 or 0.476, showing
that the resulting calculated distance in the E·I complex is
consistent with that estimated from a docked model. However, even
making the extremely unlikely assumption that
2 in the
E*I* complex has its upper limit value of 4.0 (corresponding to the two
fluorophores being held rigidly parallel to one another with
aligned dipoles) leads to an upper limit fluorophore distance of only
42.6 Å, well below the value demanded by the fully inserted model.
Although our results appear incompatible with the crystal structure of
the trypsin*antitrypsin* complex, both sets of results, as well
as the apparently contradictory results of others (15, 16), can be
rationalized within the general mechanism for serpin·proteinase complex formation that we propose in Fig.
6, which is based in part on the recent
results of Gooptu et al. (19) (see below). The
important features of this mechanism are that: (a) partial insertion of the RCL, involving substantial structural change, occurs
with the P1-P1' peptide linkage intact; (b) acyl enzyme formation occurs following partial insertion and is not concomitant with full insertion; and (c) the fully inserted form of the
acyl enzyme, E*I*2 is formed reversibly from the partially
inserted form E*I*1, and differences in serpin structure
are capable of affecting both the equilibrium position between these
two states and the activation energy barrier for state-to-state
interconversion. Here it should be noted that there is long-standing
evidence for the reversibility of E*I* formation from E·I (42-44),
although the overall equilibrium generally favors the acyl enzyme
form.

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Fig. 6.
General mechanism for E*I*
formation. Shown is a general mechanism for E*I* formation applied
to Chtr*(gray)ACT*(yellow), which accounts for
the results obtained for a variety of serpin-proteinase stable acyl
enzyme complexes (see "Discussion"). In the structures
shown, generated by QUANTA, the MCM group bound to the P1' residue of
ACT is depicted as a blue triangle and the DEACA group bound
to the amino terminus of -Chtr as a red oval. The
structure of the encounter complex is taken from the docked model of
Katz and Christianson (39) in which the RCL (in red) shows
no preinsertion into the A -sheet. The carboxyl-terminal portion of
helix F (in blue) is exposed to solvent. E·I is converted
via three or more steps (23) to EIc, in which the
RCL is partially inserted between the s3A and s5A strands, with full
insertion blocked by insertion of the carboxyl terminus of helix F, and
the P1-P1' bond is intact. The structure of the serpin portion of
E·Ic is based on the structure determined for the
-conformation of ACT (19). Residues 353-357 (P6-P2) are shown as
part of a continuous RCL, although they are not visible in the
determined structure. Conversion of E·Ic to the acyl
enzyme E*I*1 is accomplished without major conformational
change, with enzyme and P1' remaining in proximity (Table III).
Conversion of E*I*1 to E*I*2 proceeds
reversibly and results in translocation of the proteinase across the
length of serpin, accompanied by a large separation between enzyme and
P1', with helix F displaced from -sheet A by the RCL. The Chtr and
ACT portions of E*I*2 are based on the determined
structures of -Chtr (51) and cleaved ACT (52), respectively. The
latter structure shows no observed density for the segment P1' to P6'.
In the E*I*2 structure shown, the probe is placed on the
first observed residue in the cleaved structure (P7'). In the
determined structure of cleaved antitrypsin (53), which clearly shows
the P1' to P6' region, this portion of the RCL is extended even further
away from the body of the serpin.
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Feature a is supported by both time-resolved and structural
studies. In our own work, both current and previous (5, 22, 23), on the
ACT/Chtr interaction, we have shown that large fluorescent changes,
from probes placed at the P7, P13, and P1' positions in ACT precede
E*I* formation. Similar conclusions from time-resolved studies have
been reported for the interactions of antithrombin (AT) and thrombin
(14) and antitrypsin and elastase (45). In addition, structural studies
have indicated that the RCL of intact serpins can insert either
partially (19, 46) or fully into the A
-sheet (13, 47), the latter
yielding the latent serpin. In the proposed mechanism, complexation
with enzyme would favor partial insertion of the intact RCL in forming
EIc.
Feature b is a consequence of the present work, which
unequivocally shows that acyl enzyme forms in the reaction of ACT and Chtr without full insertion of the RCL. This result differs from that
of Stratikos and Gettins (15), who, employing FRET measurements from
tryptophans within trypsin-to-dansyl groups placed at different specific positions within antitrypsin, demonstrated that enzyme was
proximal to the bottom of the serpin as in E*I*2, in
agreement with the crystal structure of the antitrypsin·trypsin
complex (11). Moreover, earlier work by Stratikos and Gettins (48) showed that such insertion occurs reasonably rapidly. Similarly, Fa
et al. (16), using donor-donor energy migration and several fluorescently labeled double Cys variants of PAI-1, showed that, within
the PAI-1·u-PA complex, the P3 position (amino acid 344) was far
removed from the P1' position (amino acid 347) at the top of the serpin
but was proximal to amino acid 313 at the bottom of the serpin; in
these studies, however, there was no clear indication of how quickly P3
separates from P1'.
Feature c rationalizes the accumulation of E*I*1
in the interaction of ACT and Chtr, as opposed to the accumulation of
E*I*2 in the antitrypsin/trypsin and PAI-1/u-PA
interactions, if it is assumed that the E*I*1 complex has a
higher relative stability for the ACT:Chtr pair. A structural basis for
this assumption is provided by the crystal structure of the so-called
-conformation of the naturally occurring L55P variant of intact ACT,
as recently determined by Gooptu et al. (19). In this
structure, the space between the s3A and s5A strands is filled by
partial insertion of residues from the RCL, which bends out of the A
sheet at P12 and turns to join s1C, and by residues 164-172, which are
part of helix F-s3A turn in the native structure. We used the
-conformation as a model for the serpin portion of both
EIc and E*I*1, although recognizing
that the structures of these two species could well differ in detail.
In rationalizing the
-conformation, which has not yet been found for
any other serpin, Gooptu et al. (19) point out the high
homology between residues 164-172 and residues P9-P1, which occupy the
same positions in cleaved ACT (Table IV).
Importantly, other serpins, in particular, antitrypsin and PAI-1, show
considerably lower homology between these two sequences, consistent
with the assumption of higher relative stability for ACT and, by
extension, for Chtr*ACT*1. Whether such higher stability is
sufficient to favor Chtr*ACT*1 thermodynamically over
Chtr*ACT*2 or just increases the kinetic barrier for
conversion of Chtr*ACT*1 to a more stable Chtr*ACT*2 is unknown. Physiologically it may not matter,
because such conversion does not take place on heating
Chtr*ACT*1 at 25 or 40 °C for 15 min, and
serpin·proteinase complexes are cleared from the bloodstream rather
rapidly, with a t1/2 of 12 min observed for the
clearance of Chtr*ACT* (49, 50).
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Table IV
Sequence comparisons
A structure-based sequence comparison between the RCL from residues P9
to P1 and the carboxyl-terminal portion of helix F for four inhibitory
serpins is shown. This portion of helix F is seen to insert into
-sheet A in L55P-ACT (19).
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Finally, feature c also rationalizes the apparent conflict
in the results of Fa et al. (16), discussed above, and those of Bijnens et al. (17), working with an essentially
identical serpin-proteinase pair. The latter workers report that
monoclonal antibodies binding to an epitope comprising residues
128-131 in helix F and K154 in the turn connecting helix F
to s3A, at the bottom of the serpin, bind equally well to active PAI-1
and to the PAI-1·t-PA complex. They consider this result incompatible
with full insertion of the RCL, as in E*I*2, in which
enzyme at the bottom of the serpin would block access to epitope, but
it would be compatible with E*I*1. A resolution of this
apparent conflict is provided by the assumption that the monoclonal
antibody binds only to E*I*1, because in this case antibody
addition could shift an E*I*1/E*I*2 equilibrium distribution from dominant E*I*2 to dominant
E*I*1. A similar explanation would account for the results
of Picard et al. (18), who showed that a monoclonal antibody
recognizing residues 366-370 (P28-P24) in s5A in AT binds to the
AT·thrombin and AT·Factor Xa complexes, as well as to the complex
formed between AT and a hexapeptide corresponding to residues P14-P9,
but does not bind to native, heparin-activated, latent, or cleaved
AT.