Formation of the Covalent Chymotrypsin·Antichymotrypsin Complex Involves No Large-scale Movement of the Enzyme*

Kevin M. O'Malley and Barry S. CoopermanDagger

From the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323

Received for publication, September 15, 2000, and in revised form, October 10, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha 1-Antichymotrypsin 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. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser195 of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta -sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha 1-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E·I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha 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 beta  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 alpha -ACT, derivatized with a fluorescence donor at the P1' position, and delta -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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Bovine N-p-tosyl-L-leucine chloromethyl ketone-treated delta -chymotrypsin, alpha -chymotrypsinogen, all chromophoric proteinase substrates, N-alpha -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 right-arrow 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 epsilon 330 = 13,000 M-1 cm-1. Total protein concentration was determined using epsilon 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 delta -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 epsilon 432 = 44,000 M-1 cm-1 (24). The epsilon 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 delta -CT and DEACA-delta -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-delta -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 delta -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 (alpha -chain) from the rest of the molecule (27, 28). delta -Chtr or DEACA-delta -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 alpha -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-delta -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 lambda D1 and AEM is the absorption of all species at the emission wavelength (lambda D2). Such corrections varied from 3 to 19% for both acceptor and donor fluorescence (see below).


F=F<SUB><UP>obsd</UP></SUB>×10<SUP>−(A<SUB><UP>EX</UP></SUB><UP>+</UP>A<SUB><UP>EM</UP></SUB>)/2</SUP> (Eq. 1)

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 (lambda <UP><SUB><IT>D</IT>1</SUB><SUP><IT>A</IT>2</SUP></UP> 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(lambda D1) and AA(lambda D1) are the absorbances of the donor and acceptor, respectively, at an excitation wavelength (lambda D1) of 330 nm; and fa (equal to 0.75) is the fraction of labeled acceptor protein.
E=(1/f<SUB>a</SUB>)[A<SUB>A</SUB>(&lgr;<SUB>D1</SUB>)/A<SUB>D</SUB>(&lgr;<SUB>D1</SUB>)][F<SUB>DA</SUB>&lgr;<SUP>A2</SUP><SUB>D1</SUB>)/F<SUB>A</SUB>(&lgr;<SUP>A2</SUP><SUB>D1</SUB>)−1] (Eq. 2)

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 (lambda <UP><SUB><IT>D</IT>1</SUB><SUP><IT>D</IT>2</SUP></UP> = 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.
E=(1/f<SUB>a</SUB>)[1−F<SUB>DA</SUB>(&lgr;<SUP>D2</SUP><SUB>D1</SUB>)/F<SUB>D</SUB>(&lgr;<SUP>D2</SUP><SUB>D1</SUB>)] (Eq. 3)

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.


R<SUP>6</SUP><SUB>o</SUB>=8.8×10<SUP>−5</SUP>(&kgr;<SUP>2</SUP>n<SUP>−4</SUP>&phgr;<SUB>d</SUB>J) (Eq. 4)
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); phi 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 kappa 2 is the orientation factor, describing the relative orientation in space of the transition dipoles of the donor and acceptor. kappa 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 (kappa 2 = 2/3) or 21.0Å (kappa 2 = 0.476).

Modeling-- Modeling of the MCM-S359C-rACT·DEACA-delta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site of DEACA Labeling of delta -Chtr-- The site or sites of labeling within delta -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 alpha -chain (residues 1-13) and, by inference, to the only amino group within this chain, the alpha -amino group of Cys1. Accordingly, preparations containing 0.75 DEACA per delta -Chtr were used in all kinetics experiments.

Direct determination of the stoichiometry of alpha -chain labeling was provided by RP-HPLC analysis of the soluble fraction of a performic acid oxidized sample of DEACA-delta -Chtr (Fig. 1) as described under "Experimental Procedures." An analysis of the 75% labeled DEACA-delta -Chtr gave a labeled alpha -peptide/unlabeled alpha -peptide ratio of 3:1, measured by corrected peak area at A215. The assignment of peak 2 as the DEACA-labeled alpha -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 alpha -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-delta -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 alpha -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.

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-delta -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-delta -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 delta -Chtr.


                              
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Table I
Functional properties of modified ACT and Chtr

Fluorescence Spectra of DEACA-delta -Chtr, MCM-S359C-rACT, and the DEACA-delta -Chtr*MCM-S359C-rACT* Complex-- Relative to the sum of the contributions of single-labeled complexes DEACA-delta -Chtr*wt-rACT* and delta -Chtr*MCM-S359C-rACT*, excitation at 330 nm of the DEACA-delta -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-delta -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-delta -Chtr*MCM-S359C-rACT*; b, delta -Chtr*MCM-S359C-rACT*; c, DEACA-delta -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.

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-delta -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 delta -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-delta -Chtr. Upper trace, relative fluorescence on adding MCM-S359C-rACT to delta -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 delta -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-delta -Chtr with excitation at 330 nm (solid trace); solid circles represent the fractional conversion to MCM-S359C-rACT*DEACA-delta -Chtr*, following the addition of MCM-S359C-rACT to DEACA-delta -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-delta -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.

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.
<UP>E · I</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB></UL></LIM> <UP>EI<SUB>a</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>EI<SUB>b</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>E*I*</UP>

<UP><SC>Scheme</SC> 1</UP>
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-delta -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.


                              
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Table II
Rate constants (5 °C, pH 7.0)

Rate of DEACA-delta -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-delta -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-delta -Chtr. A, changes observed at 475 nm upon the addition of MCM-S359C-rACT to DEACA-delta -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-delta -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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Table III
Calculated efficiencies and distances

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 alpha -amino group in alpha -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 alpha -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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


<UP>E · I</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB></UL></LIM> <UP>EI<SUB>a</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>EI<SUB>b</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>EI<SUB>c</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>4</SUB></UL></LIM> <UP>E*I*</UP>

<UP><SC>Scheme</SC> 2</UP>
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 beta -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 kappa 2 in Equation 4. Above (see "Experimental Procedures"), we have provided the rationale for using kappa 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 kappa 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 delta -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 beta -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 delta -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 beta -sheet A by the RCL. The Chtr and ACT portions of E*I*2 are based on the determined structures of alpha -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.

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 beta -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 delta -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 delta -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 delta -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 beta -sheet A in L55P-ACT (19).

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.


    ACKNOWLEDGEMENTS

We thank Nora Zuño for excellent technical assistance; Drs. Xuzhou Liu and Harvey Rubin for advice and training in molecular biology techniques; Dr. James A. Huntington for making the coordinates of the trypsin*antitrypsin* complex structure available prior to publication; and Dr. Michael Therien for the use of the spectrofluorometer.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant AG 10599.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323. Tel.: 215-898-6330; Fax: 215-898-2037; E-mail: cooprman@pobox.upenn.edu.

Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M008478200


    ABBREVIATIONS

The abbreviations used are: ACT, alpha 1-antichymotrypsin; rACT, recombinant alpha 1-antichymotrypsin; AT, antithrombin; BMMC, 4-bromomethyl-7-methoxycoumarin; Chtr, alpha -chymotrypsin; delta -Chtr, delta -chymotrypsin; DEACA, 7-diethylaminocoumarin-3-carboxylic acid; FRET, fluorescence resonance energy transfer; HPLC, high pressure liquid chromatography; MCM, (7-methoxycoumaryl-4)-methyl; PAGE, polyacrylamide gel electrophoresis; PAI-1, plasminogen activator inhibitor-1; RCL, reactive center loop; serpin, serine proteinase inhibitor; SI, stoichiometry of inhibition; TLCK, N-alpha -tosyl-L-lysine chloromethyl ketone; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase HPLC.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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