Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323
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ABSTRACT |
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Serpins, serine proteinase inhibitors, form
enzymatically inactive, 1:1 complexes (denoted E*I*) with
their target proteinases, that only slowly release I*, in which the
P1-P1' linkage is cleaved. Recently we presented evidence that the
serpin antichymotrypsin (ACT, I) reacts with the serine proteinase
chymotrypsin (Chtr, E) to form an E*I* complex
via a three-step mechanism, E + I E ·I
EI'
E*I* in which EI',
which retains the P1-P1' linkage, is formed in a partly or largely
rate-determining step, depending on temperature (O'Malley, K. H, Nair,
S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354-5359). Here we extend these studies
through the introduction of a new assay for the formation of the
postcomplex fragment, corresponding to ACT residues 359 (the P1'
residue) to 398 (the C terminus), coupled with rapid quench flow
kinetic analysis. We show that the E·I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex
fragment with the same rate constant, so that both species arise from
EI' conversion to E*I*. These results support
our earlier conclusion that the P1-P1' linkage is preserved in
EI' and imply that E*I* corresponds to a
covalent adduct of E and I, either acyl enzyme or the
tetrahedral intermediate formed by water attack on acyl enzyme.
Furthermore, we show that the A347R (P12) variant of rACT, which is a
substrate rather than an inhibitor of Chtr, has a rate constant for
postcomplex fragment formation from the E·I complex very
similar to that observed for WT-rACT, implying that EI' is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a
proposed scheme for ACT interaction with Chtr that is considered in the
light of relevant results from studies of other serpin-serine proteinase pairs.
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INTRODUCTION |
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Antichymotrypsin (ACT, I)1 is a human serine proteinase inhibitor (serpin), 398 amino acids long, that, as is typical of serpins (1, 2), forms an enzymatically inactive, 1:1 complex (denoted E*I*) with its target proteinases, releasing free enzyme (E) and cleaved ACT (I*) only very slowly (3, 4). The involvement of ACT in Alzheimer's disease (5, 6) and in the regulation of the inflammatory response (7) as well as of prostate-specific antigen activity (8), makes it a particularly interesting protein for study. In the interaction of ACT with chymotrypsin (Chtr) to form an E*I* complex, both proteins undergo extensive conformational change (4, 9); the nonlability of E*I* may be due either to distortion of the enzyme active site within the complex (4, 9) 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," which in intact
I extends out from the rest of the molecule, contains a segment of
modified -helix (10) and is the primary interaction site between the
inhibitor and the target proteinase. Following standard nomenclature
(11), these residues are designated P1 and P1'. In released I* residues
P1-P14 are inserted into
-sheet A, the dominant structural element
in ACT, as strand 4A (s4A). The
-sheet C is also reinforced, with
the result that the P1 and P1' residues are separated by 70 Å (12).
Recent fluorescence energy transfer studies suggest substantial, if not
complete, s4A insertion within E*I* for the serpin:serine
proteinase pairs
1-proteinase inhibitor:trypsin (13) and
plasminogen activator inhibitor-1:u-plasminogen activator (14).
E*I*s formed from a variety of serpin-serine proteinase
pairs are stable to both boiling water and SDS treatment, implying covalent bond formation between enzyme and serpin. Parallel studies by
both Lawrence et al. (15) and Wilcynska et al.
(16) have clearly shown that the P1-P1' bond is cleaved within the
complexes formed by several such pairs (including plasminogen activator inhibitor-1:t-plasminogen activator, plasminogen activator
inhibitor-1:u-plasminogen activator, 1-proteinase
inhibitor:human neutrophil elastase,
1-proteinase
inhibitor: porcine pancreatic elastase and
2-antiplasmin:plasmin), providing strong evidence that
E*I* corresponds either to an acyl enzyme, or, less likely
given the stability of E*I*, to the tetrahedral intermediate
formed by water attack on the acyl enzyme (17).
Although full inhibition of Chtr requires only 1 eq 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 (18) and even with Chtr, some mutations within the reactive center loop yield inhibitors with SI > 1 (e.g. SI is 2.4 for the L358R variant) (3). In the extreme, some variants, such as A347R (P12) and T345R (P14), in which small, neutral side chains within the "hinge" region of rACT are replaced by the charged, bulky arginine side chain, show no inhibition of Chtr (SIs > 100) and no E*I* formation, but are cleaved by Chtr in the reactive center loop with complete (A347R) or almost complete (T345R) insertion of s4A (19, 20). These results, which parallel results obtained with other serpins (21, 22), have been interpreted as indicating that on interaction of serpins and serine proteinases, there is a partitioning between substrate and inhibitor pathways, raising the following questions. First, what is the sequence of events leading to E*I* formation? Second, where within the sequence does partitioning occur?
Recently we presented evidence for a minimal scheme (Scheme
1) for the interaction of rACT and Chtr
leading to the formation of E*I* (23). The main features of
this scheme were that: (a) there was at least one
intermediate between the encounter complex, E·I, and
E*I*; (b) conversion of E·I to
EI' involved at least partial reactive center loop insertion
into -sheet A of rACT, as shown by a blue-shifted increase in
fluorescence intensity when a fluorescent derivative of rACT (at
position P7 within the reactive center loop) was reacted with Chtr;
(c) the P1-P1' linkage was preserved in EI', as
inferred from the fact intact inhibitor was released on rapid acid
denaturation of EI'; and (d)
k3
k2 at 25 °C,
but is similar in magnitude at 40 °C. We also speculated that
EI', rather than E·I, was the common
intermediate from which partitioning between the inhibitor and
substrate pathways occurred.
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Here we extend these studies by introducing a new assay for quantitatively measuring the appearance of the so-called postcomplex fragment (16), formed as a result of P1-P1' cleavage and corresponding to ACT C-terminal residues 359-398, and use this assay to measure the first-order rate constant for postcomplex fragment formation, at saturating enzyme concentration, both for WT-rACT and for the A347R variant of WT-rACT.
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EXPERIMENTAL PROCEDURES |
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Materials--
rACT and A347R-rACT were obtained as described
earlier (20, 24). Bovine
(N-p-tosyl-L-lysine
chloromethyl ketone-treated) Chtr was from Calbiochem. rACT and Chtr
concentrations were determined as described earlier (23, 24). The
concentration of A347R-rACT was determined assuming an
A2801% of 8.0 cm
1
(19). The chromophoric proteinase substrate SucAAPF-pNA,
phenylmethylsulfonyl fluoride and bovine serum albumin were obtained
from Sigma. Standard proteins for SDS-PAGE gel calibration were from
Bio-Rad. HPLC-grade acetonitrile was from Fisher, and HPLC-grade
trifluoroacetic acid was from Pierce (sequanal grade).
Rapid Kinetic Studies-- Rapid quenched flow kinetic studies were carried out using a KinTek Chemical-Quench flow model RQF-3 machine as described earlier (23). Quenching was achieved with 0.1 M HCl.
In preparing samples for HPLC analysis and quantification of rACT C-terminal peptide formation, the quenched rACT:Chtr sample (85 µl) was treated with 12 M urea (118 µl) and 300 mM phenylmethylsulfonyl fluoride (2 µl) and incubated for 1 h at 25 °C. Following centrifugation at 12,000 rpm in an Eppendorf centrifuge (no. 5414) for 2 min, 200 µl of supernatant was injected into a Perkin-Elmer Series 4 system, equipped with an LC-95 (Perkin-Elmer, 4.5 µl flow cell) variable wavelength detector, using a Rainin C-18 column (Microsorb-MVTM, 50 × 4.6 mm, 300 Å, 5 µm, 0.7 ml/min). HPLC data were analyzed using Turbochrom Navigator software from Perkin-Elmer. A215 values were used to quantitatively estimate the eluted peptide (25).Other Methods-- Quenched rACT:Chtr samples were analyzed on SDS-PAGE, and stained protein bands were quantified, as described earlier (23). Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on a VG Tofspec linear time-of-flight mass spectrometer (Fisons Instruments, Danver, MA) at the Protein Chemistry Laboratory in the Medical School of the University of Pennsylvania.
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RESULTS |
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Purification and Quantification of Postcomplex Fragment-- Treatment of rACT with Chtr leads to postcomplex fragment formation. Following denaturation of the sample with dilute HCl and concentrated urea, to ensure dissociation of the postcomplex fragment from the remainder of the rACT molecule, the peptide was purified from other components of the reaction mixture by RP-HPLC, as shown in Fig. 1A. Its retention time was close to the value predicted for amino acids 359-398 based on the use of Rekker's constants for the constituent amino acids (26). Proof that the indicated postcomplex fragment peak contains the C-terminal peptide was provided by MALDI-MS analysis, which gave a mass of 4624 (for Ala2Arg3Asn3AspGln2GluIle4Leu2Lys2Met2Phe4Pro3Ser2Thr5Val4, the theoretical M + H+ is 4623.5). In simple preparative experiments, the yield of recovered postcomplex fragment, calculated from HPLC peak area as described previously (25), was found to be 85 ± 5%. The difference from 100% is attributable to losses on the RP-HPLC column.
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Rate of Postcomplex Fragment Release from the rACT·Chtr
Complex--
Rate constants for postcomplex fragment release following
mixing of rACT and excess Chtr were measured using a quenched stopped flow apparatus, and determining the amount of released fragment (Fig.
1B) as a function of incubation time prior to the quench. The yield of recovered peptide was 75 ± 9%. This value, slightly lower than that above, reflects losses in the rapid quench apparatus in
addition to those on the RP-HPLC column. Rates were measured under
strictly first-order conditions, as demonstrated in Fig. 2 (upper panel). Earlier we
showed that quantitative scanning of stained SDS-PAGE analyses of
quenched reaction mixtures, in which E*I* is well resolved
from both E and I, could also be used to determine rate
constants for the formation of the SDS-stable complex E*I*
(23). The results displayed in Fig. 2 (lower panel) demonstrate that formation of both E*I* and postcomplex
fragment proceed at the same rate at pH 7.5 and 40 °C. Rate
constants for the two processes were also identical for experiments
performed at both pH 7.5 and 9.0 at 25 °C within experimental error,
as summarized in Table I. Experiments
nos. 1 and 2 make clear the independence of the measured first-order
rate constant on [Chtr] in the concentration range employed,
demonstrating that this constant (7 ± 1 s1 at
25 °C, pH 7.5) is for reaction of the rACT·Chtr complex.
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Rate of Postcomplex Fragment Formation from the A347R-rACT·Chtr
Complex--
Proceeding as above, the first-order rate constant for
postcomplex fragment formation was measured for the reaction of the substrate A347R-ACT with excess Chtr, giving the results shown in Table
I. The value obtained (3.9 ± 0.7 s1 at 25 °C, pH
7.5) is 60% of that for rACT reaction with Chtr, and, as above, its
lack of dependence on [Chtr] demonstrates that it measures reaction
of the A347R-rACT·Chtr complex.
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DISCUSSION |
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The postcomplex fragment assay we introduce in this report, along
with the assay for E*I* formation we described earlier (23) based on SDS-PAGE analyses, provide two new general methods for monitoring serpin-proteinase interactions. In contrast, earlier demonstrations that the P1-P1' bond is cleaved within several E*I* complexes relied on assay of the newly formed
-NH2 terminus at P1' (15, 16), an approach only
applicable in the absence of lysines in the postcomplex fragment.
Coupling these new assays to a rapid quench flow kinetic analysis, we
provide the first demonstration that the overall first-order rate
constants for both postcomplex fragment and E*I* formation
from the E·I encounter complex are indistinguishable from
one another over a range of experimental conditions, including at
40 °C when k2 is similar in magnitude to
k3. Thus, within the context of Scheme 1,
postcomplex fragment formation must accompany EI' conversion
to E*I*, supporting our earlier conclusion that the P1-P1'
linkage is preserved in EI'.
The second major result of this work is the demonstration that postcomplex fragment formation from Chtr complexes of both the inhibitor WT-rACT and the substrate A347R-rACT proceed with very similar first-order rate constants. This result straightforwardly supports the hypothesis that 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 a step that is largely rate-determining, i.e. EI'. By contrast, partitioning from E·I rather than from EI' would require that two quite different transformations, conformational change of E·I to yield EI' and hydrolysis of I within E·I to yield E + Is, coincidentally proceed with very similar rate constants, which we consider unlikely.
If partitioning between the inhibitor and substrate pathways is from EI', then the putative partial insertion accompanying EI' formation must be insufficient to distort the catalytic machinery of the proteinase. As a result, further conformational change in the serpin-proteinase complex accompanies EI' to E*I* conversion, and it is this further change that must be coupled to proteinase distortion and stabilization of E*I* toward hydrolysis.
Is Scheme 1 generally applicable for serpin-serine proteinase
interactions? Scheme 1 is similar to those proposed by Olson et
al. (27) and Stone and Le Bonniec (28) in positing that conversion
of the E·I encounter complex to E*I* proceeds
via an intermediate, EI', and that the reactive center loop
is at least partially inserted into -sheet A in EI'. Also
noteworthy is the similarity in values of the overall first-order rate
constants for E·I conversion to E*I* in this
work (7 s
1 for rACT and Chtr at 25 °C and pH 7.5) and
in reactions of the heparin complex of antithrombin and thrombin (5 s
1) (29) and of t-plasminogen activator and two different
reactive center loop fluorescent derivatives of plasminogen activator
inhibitor-1, at P1' (8 s
1) and P9 (4 s
1)
(15), under similar conditions (25 °C and pH 7.4).
However, the schemes proposed by Olson et al. (27) and Stone
and Le Bonniec (28) differ from one another in two significant respects. The first concerns the structure of EI'. Olson
et al. propose that EI' involves a covalent bond
(either tetrahedral intermediate or acyl enzyme) between the active
site Ser-195 and the serpin, based mainly on the lack of fluorescence
change following anhydrotrypsin (i.e. with dehydroalanine in
place of Ser-195) complex formation with NBD-labeled P9 Ser Cys
plasminogen activator inhibitor-1. In contrast, complexation of trypsin
with NBD-labeled P9 Ser
Cys plasminogen activator inhibitor-1
results in a large fluorescence enhancement. On the other hand, Stone
and Le Bonniec propose that EI' does not involve covalent
bond formation, based on their demonstration that, in reacting with the
heparin complex of antithrombin, both WT thrombin and its S195A variant
share a common pathway through EI' formation, with identical
values for k2, although only WT thrombin can
complete the reaction to form E*I*. In their scheme (as in
ours) k2 is largely rate-determining for
E*I* formation. A possible reason for this difference is
that dehydroalanine substitution for Ser-195 might be more disruptive than Ala-195 substitution, effectively blocking E·I
conversion to EI'. Alternatively, different serpin-serine
proteinase pairs may simply react via different pathways. Our present
results are compatible either with no covalent interaction between
E and I in EI' or with formation of the initial
tetrahedral intermediate on Ser-195 attack on the P1 carbonyl, and thus
shed no further light on this issue. The second point of difference
concerns the species from which partitioning into substrate and
inhibitor pathways occurs, Olson et al. placing it at
EI', as in Scheme 1 and Stone and Le Bonniec placing it at
E·I, although in neither case is the supporting evidence
strong. As discussed above, our results support EI' as the
species from which partitioning occurs.
We conclude that Scheme 1, in which (a) formation of EI' from E·I is at least partly rate-determining in overall E*I* formation from the E·I encounter complex, (b) the P1-P1' linkage is preserved in EI', (c) partitioning between substrate and inhibitor pathways occurs from EI', and (d) E*I* corresponds to a covalent adduct of E and I, most likely acyl enzyme, is likely to provide a general, minimal description of serpin-serine proteinase interaction, but that whether there is covalent bond formation between E and I in EI' might vary from system to system.
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ACKNOWLEDGEMENTS |
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We acknowledge with thanks the excellent technical assistance in several aspects of this work of Nora Zuño, and the participation of Dr. Kui Xu in the development of the postcomplex fragment assay.
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FOOTNOTES |
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* 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.
Current address: EPIX Medical Inc., 71 Rogers St., Cambridge, MA
02142.
§ To whom correspondence should be addressed. Tel.: 215-898-6330; Fax: 215-898-2037; E-mail: cooprman{at}pobox.upenn.edu.
1
The abbreviations used are: ACT,
1-antichymotrypsin; BSA, bovine serum albumin; Chtr,
-chymotrypsin; HPLC, high performance liquid chromatography;
MALDI-MS, matrix-assisted laser desorption ionization mass
spectrometry; NBD,
N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine; PAGE, polyacrylamide gel electrophoresis; rACT, recombinant ACT; RP,
reverse phase; serpin, serine proteinase inhibitor; SI, stoichiometry of inhibition; WT, wild type.
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REFERENCES |
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