(Received for publication, August 6, 1996, and in revised form, October 29, 1996)
From the Departments of Chemistry and
§ Medicine, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6323
Serine proteinase inhibitors (serpins) form
enzymatically inactive, 1:1 complexes (denoted E*I*) with
their target proteinases that release free enzyme and cleaved inhibitor
only very slowly. The mechanism of E*I* formation is
incompletely understood and continues to be a source of controversy.
Kinetic evidence exists that formation of E*I* proceeds via
a Michaelis complex (E·I) and so involves at least two
steps. In this paper, we determine the rate of E*I*
formation from -chymotrypsin and
1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of
-chymotrypsin with a fluorescent derivative of
1-antichymotrypsin (derivatized at position P7 of the
reactive center loop); and second, by a rapid mixing/quench approach
and SDS-polyacrylamide gel electrophoresis analysis. In some cases,
serpins are both substrates and inhibitors of the same enzyme. Our
results indicate the presence of an intermediate between
E·I and E*I* and suggest that the
partitioning step between inhibitor and substrate pathways precedes
P1-P1
cleavage.
1-Antichymotrypsin
(ACT)1 is a human serine proteinase
inhibitor (serpin), a superfamily of proteins believed to have evolved from a common ancestral gene over ~500 million years (1-3). The involvement of ACT in Alzheimer's disease (4, 5) and in the regulation
of the inflammatory response (6)2 as well
as of prostate-specific antigen activity (8) makes it a particularly
interesting protein for study. As is typical of serpins, ACT (I) forms
an enzymatically inactive, 1:1 complex (denoted E*I*) with
its target proteinases (for example, chymotrypsin) that releases free
enzyme and cleaved ACT (I*) only very slowly (9, 10). The complex is
designated E*I* to indicate that conformational change has
taken place in both the enzyme (10, 11) and inhibitor (12, 13)
moieties. For some serpin-proteinase interactions, full inhibition of
proteinase requires >1 eq of serpin. The ratio of moles of inhibitor
required per mole of proteinase for 100% inhibition is defined as the
stoichiometry of inhibition (SI). Values of SI > 1 reflect a
partitioning of serpin between inhibitor and substrate pathways, giving
rise to "suicide inhibitor"-type kinetics (9, 14-16).
A characteristic feature of the serpin-proteinase complex is that it is
stable to both heat and SDS treatment, implying covalent bond formation
between enzyme and serpin. The nonlability of E*I* may be
due either to distortion of the enzyme active site within the complex
(10, 11) or to inaccessibility of the covalent E-I linkage
toward attacking nucleophilic water, or both. The position of cleavage
in released I* occurs within a so-called "reactive center loop,"
which in intact I extends out from the rest of the molecule, contains a
segment of modified -helix (17-21), and is the primary interaction
site between the inhibitor and the target proteinase. Following
standard nomenclature (22), the position of cleavage takes place
between the P1 and P1
sites of the inhibitor, which in ACT corresponds
to positions 358 and 359. Residues proceeding toward the N and C
termini from P1 and P1
, respectively, are labeled with higher P and P
numbers. The reactive center loop extends from approximately P17 to
P9
. Cleavage of intact I to form I* is accompanied by a large decrease
in free energy and a substantial gain in stability toward denaturation by either heating or denaturing agents (23, 24). In I*, residues P1-P14 are inserted into
-sheet A, the dominant structural element in ACT, as strand 4A.
-Sheet C is also reinforced, with the result that the P1 and P1
residues are separated by 70 Å (25).
Kinetic evidence has existed for some time that formation of the nonlabile serpin-proteinase complex involves at least two steps (26-28): the second-order association of E and I in an "encounter" or Michaelis complex (E·I), followed by its conversion, in a first-order process, to E*I* (Scheme 1).
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Formation of the E*I* complex requires completion of a
minimum of three processes: 1) establishment of P1 interaction with the
S1-binding site of the enzyme and of perhaps subsite interactions as
well, necessitating at least partial unwinding of the helical portion
of the reactive center loop; 2) partial or full insertion of strand 4A
into -sheet A (18, 36); and 3) P1-P1
cleavage and formation of the
covalent acyl-enzyme (or tetrahedral intermediate). However, the
relative timing of these processes is unknown.
In this paper, we determine the rate of E*I* formation from
E·I using two approaches. In the first, we derivatize the
A352C-rACT variant (at position P7) with the fluorescent reagent
4-bromomethyl-7-methoxycoumarin (BMMC), demonstrate that the resulting
(7-methoxycoumaryl-4)-methyl derivative of A352C-rACT (MCM-A352C-rACT)
inhibits -chymotrypsin in the normal manner, and measure its rate of
complex formation with
-chymotrypsin by stopped-flow
spectrofluorometry. In the second, we apply a rapid mixing/quench
approach and SDS-PAGE analysis to determine the rate of E*I*
formation on reaction of either MCM-A352C-rACT or rACT with
-chymotrypsin. Our results indicate the presence of an intermediate
between E·I and E*I* and suggest that the
partitioning step between inhibitor and substrate pathways precedes
P1-P1
cleavage.
Bovine
N-p-tosyl-L-leucine
chloromethyl ketone-treated chymotrypsin and human neutrophil elastase
were obtained from Calbiochem or Sigma. The concentrations of these
enzymes and of rACT were determined as described earlier (16). All
chromophoric proteinase substrates, dithiothreitol, and
phenylmethylsulfonyl fluoride were obtained from Sigma. BMMC was
acquired from Molecular Probes, Inc. (Eugene, OR). SDS-PAGE analysis
was performed according to Laemmli (37). Standard proteins were from
Bio-Rad.
A352C-rACT was constructed using sequence overlap
expression polymerase chain reaction (38, 39) and the ACT expression vector described previously (16, 40). The internal primers coding for
the Ala Cys mutation are as follows:
5
-GCCACATGCGTCAAAATCACCCTCC-3
and
5
-GATTTTGACGCATGTGGCAGCAGATGCT-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. A352C-rACT and rACT were
purified to homogeneity as described earlier (9, 16, 41).
A352C-rACT (2 µM in 50 mM Tris and 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. The protein was then concentrated using an Amicon concentrator; diluted 10-fold in 2 M (NH4)2SO4 and 100 mM sodium phosphate, pH 7.0; and purified by hydrophobic interaction chromatography using a procedure similar to that of Kvassman and Shore (42) for the separation of active from latent plasminogen activator inhibitor-1. Application of the diluted sample onto a Synchropak propyl column (250 × 7.8 mm; SynChrom, Inc.) was followed by washing with 1 void volume of 1.8 M (NH4)2SO4 and 100 mM sodium phosphate, pH 7.0 (Buffer A). Protein was then eluted with a two-step linear gradient of decreasing antichaotropic salt: 100 to 28% Buffer A in 15 min, with a flow rate 2 ml/min; and 28 to 0% Buffer A in 60 min, with a flow rate of 0.7 ml/min. Protein elution was monitored by A215. MCM-labeled protein elution was monitored by A330 as well. This procedure separates modified from unmodified protein. Solutions of pure inhibitor were concentrated and buffer-exchanged using Centriprep-30 and Centricon-30 concentrators (Amicon, Inc.).
Characterization of MCM-A352C-rACTThe stoichiometry of MCM
labeling was determined by absorbance measurements. Total bound MCM
concentration was determined using 330 = 13,000 cm
1 (43). Total protein concentration was determined
using
280 = 39,000 cm
1, calculated as the
sum of the coefficients of the unlabeled protein (36,000 cm
1, calculated from an ACT solution standardized by
Bradford analysis (44)) and the MCM group (3000 cm
1, as
determined by the
A280/A330 ratio for
BMMC). Purified protein had a calculated stoichiometry of 1.1 MCM/protein. Titration reactions of
-chymotrypsin activity were
performed at pH 7.5 as described (16).
Stopped-flow measurements were made and fluorescence emission spectra were acquired using an Applied Photophysics !SX.18MV stopped-flow spectrofluorometer with excitation at 330 nm. For rate measurements, emission was monitored at 400 nm. First-order rate constants were obtained by fitting a single exponential to unsmoothed traces.
Rapid Quenched FlowRapid quenched-flow kinetic studies
were carried out using a KinTek Chemical-Quench-Flow Model RQF-3
machine with an estimated dead time of 5 ms (45, 46). Quenched-flow
experiments were carried out by rapid mixing of the enzyme solution in
one sample loop with the inhibitor solution in the other and quenching
with 0.1 N HCl. SDS-PAGE analysis was performed on aliquots
of the quenched reaction mixture using gels containing 12 or 15%
polyacrylamide. Prior to analysis, all samples were treated with 1 M Tris base (2 µl/20 µl of quenched solution), to
neutralize the excess HCl present in the quenched samples, and with
phenylmethylsulfonyl fluoride at a final concentration of 2 mM, to rapidly inactivate any renatured -chymotrypsin.
Samples were boiled for 4 min prior to application to the gels. Gels
were stained for ~1 h in Coomassie Blue staining solution (0.1%
Coomassie Blue R-250 in 40% MeOH and 10% AcOH) and then destained for
12-15 h with 7.5% AcOH and 5% MeOH.
The protein bands on the
destained gels were quantified using densitometric analysis. Scanning
was performed at a protein concentration range for which band intensity
was proportional to the concentration of protein present. Gel
photographic images were stored as GRAYSCALE pictures in the ·TiFF
format and were processed using the National Institutes of Health
IMAGE program (Version 1.60) on a Macintosh computer using
National Institutes of Health soft-FPU. Sample data are presented
in Fig. 1.
Virtually complete derivatization of
A352C-rACT was achieved under the conditions used for preparing
MCM-A352C-rACT. By contrast, reaction of rACT with BMMC under exactly
the same conditions gave only minor derivatization, reflecting the low
reactivity of the single Cys residue (Cys-237) within wild-type ACT,
which is buried within -sheet B of rACT (20), as well as slow
nonspecific reaction with carboxylate residues in the protein (43).
MCM-A352C-rACT inhibits -chymotrypsin with a stoichiometry of
inhibition of ~1 (data not shown). The
MCM-A352C-rACT·
-chymotrypsin complex is stable toward SDS
denaturation, as shown by SDS-PAGE analysis (see below), in the usual
manner of serpin-proteinase complexes and is cleaved by catalytic
amounts of human neutrophil elastase, similar to what is found for
wild-type rACT.
Excitation of a solution of
MCM-A352C-rACT at 330 nm gives the emission spectrum shown in Fig.
2, with max = 399 nm. Human neutrophil
elastase-induced cleavage leads to an increase in emission intensity
and a slight blue shift (
max = 397 nm), consistent with
the expected shift to a more hydrophobic environment as the chromophore
moves from a solvent-exposed position to one that is at least partly
buried within
-sheet A. The fluorescence spectrum of the
MCM-A352C-rACT·
-chymotrypsin complex is similar to that of cleaved
MCM-A352C-rACT.
Mixing of -chymotrypsin and MCM-A352C-rACT led to an increase in
fluorescence at 400 nm, which was used to measure the rate constant for
complex formation in a stopped-flow spectrofluorometer. All
stopped-flow reactions were carried out under pseudo first-order conditions, with
-chymotrypsin (denoted E) present in
large molar excess over MCM-A352C-rACT (denoted I). Results obtained at
pH 7.0 and 40 °C are shown in Fig. 3. Rate constants
for complex formation as a function of [I], temperature, pH, and
ionic strength are presented in Table I. As predicted by
Scheme 1, at high enough [E], the measured first-order
rate constant becomes independent of [E] and reaches a
saturated value. Under our conditions, [E]
65 µM satisfies this condition.
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Our results are similar to those of Shore et al. (13), who
carried out an analogous study measuring the rate of complex formation
between a fluorescent derivative of plasminogen activator inhibitor-1
(derivatized at P9) and plasminogen activator. These workers reported a
value for kf (the rate constant determined by the
change in fluorescence) of 4 s1 (at pH 7.4 and 25 °C)
and found that the large increase in fluorescence intensity and the
13-nm blue shift accompanying complex formation were virtually
identical to the changes observed on cleavage of derivatized
plasminogen activator inhibitor-1.
An Arrhenius plot of kf at pH 7 (15-40 °C) gives a good straight line and an apparent activation energy of 19 kcal/mol, indicating a substantial energy barrier for the process leading to fluorescence change. The rate constant increases 2-fold in the pH range 6-8, but the addition of 1 M NaCl has little effect.
Rate of MCM-A352C-rACT·Monitoring the buildup of the SDS-stable complex E*I* provides another way of measuring complex formation. E*I* is stable toward 0.1 N HCl (data not shown). As a consequence, rates of E*I* formation could be measured by 0.1 N HCl quenching at various times following rapid mixing of E and I and quantitative scanning of stained SDS-PAGE analyses of the quenched reaction mixtures (Fig. 1).
The results obtained are summarized in Table I. In terms of Scheme 1, our expectation was that E·I would not survive the quench and would be measured as free E and intact I. This expectation was confirmed by the result that the observed first-order rate constant for E*I* formation (kg) saturates as a function of E concentration (Experiments 20-22). The slight downturn in rate constant at the highest enzyme concentration used (0.2 mM, Experiment 23) might be due to a nonspecific protein effect. All rate constants were determined at [E]o in excess over [I]o and could be measured by either E*I* buildup or I disappearance with similar results. E*I* buildup was chosen for calculation of rate constants because it could be measured with greater precision.
Although the saturated rates of complex formation for MCM-A352C-rACT
reaction with -chymotrypsin are not significantly different when
measured by stopped-flow spectrofluorometry or by quenched stopped flow
at 25 °C (Table I, compare Experiments 1 and 2 with Experiment 3),
at 40 °C, the stopped-flow rate is clearly faster (~2.5-fold)
(Fig. 3 and Table I, Experiments 4 and 6). This difference demonstrates
that Scheme 1 is inadequate to explain the kinetics of E*I*
formation, as discussed below. We also note that the rate constants for
E*I* formation from both rACT and MCM-A352C-rACT are similar
in magnitude and that the rate constant for rACT reaction increases
3-6-fold over the pH range 6.0-9.0 (Table I, Experiments 21, 27, and
28).
Inspection of Fig. 1 reveals a small buildup with time of cleaved I,
migrating just ahead of intact I. Such a band was observed in reactions
of both MCM-A352C-rACT and rACT with -chymotrypsin. Measurement of
these band intensities allowed us to estimate the fractions of cleaved
I formed during the course of E*I* formation, which are 0.20 and 0.17 for MCM-A352C-rACT (at pH 7.0 and 40 °C) and rACT (at pH
7.5 and 40 °C), respectively.
At 40 °C, the saturated rate of
fluorescence change on mixing of MCM-A352C-rACT with -chymotrypsin
exceeds the saturated rate of overall E*I* formation (Fig.
3). This result provides clear kinetic evidence for an intermediate
between E·I and E*I*, which, unlike
E*I*, does not survive the quench/SDS-PAGE analysis procedure. Earlier, we (9) and others (14, 15, 47, 48) proposed the
existence of an intermediate between E·I and
E*I* from which partitioning could occur between the
inhibitor pathway, leading to E*I* formation, and the
substrate pathway, leading to enzyme and cleaved inhibitor (Is)
release, to account for the finding that some serpin-proteinase pairs
have SI values > 1 (Scheme 2). For reasons of parsimony, we
equate the intermediate we demonstrate kinetically in this work with
EI
in Scheme 2.
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These simulations predict a lag in the formation of E*I*,
which we have been unable to demonstrate unequivocally, for two reasons. First, the predicted lag is most obvious over a time scale
(0-10 ms) that brackets the dead time (~5 ms) of our instrument. Making the lag more obvious by lowering the temperature is not an
option because the buildup of EI depends on the higher
temperature. Second, background in the gel lowers the precision with
which low levels of protein can be quantified.
At 40 °C, k3 (the rate constant for acylation
of -chymotrypsin within the complex) is similar in magnitude to
k2. By contrast, k3 must
be considerably larger than k2 at 25 °C,
given the similarity of kf and kq
(~7 ± 2 s
1) at this temperature. We infer that
k3 has a small activation energy between 25 and
40 °C. This is reminiscent of the temperature dependence of the rate
constants for formation of acyl-chymotrypsin from the Michaelis complex
of the enzyme with either p-nitrophenyl acetate or
p-nitrophenyltrimethyl acetate (50). These rate constants display a dramatic drop in activation energy from 21 kcal/mol (measured
from 6 to 20 °C) to 1 kcal/mol (measured from 23 to 36 °C),
reflecting a thermally induced transition (at ~25 °C) between two
forms of enzyme with different catalytic activities.
Acyl-chymotrypsin formation on reaction with model amide substrates depends on His-57 being in the neutral form. The relevant pKa within the enzyme-substrate complex varies between 6 and 7, depending on the substrate (7). The pH dependences we observe for both kq and kf are consistent with the need for a neutral His-57, although other ionizable group(s) within the E·I complex may also modulate these processes. The decrease in rates observed at lower pH suggests that clear demonstration of a lag in E*I* formation might be possible for rates measured at 40 °C below pH 6.
Structure of EIIn addition to providing evidence for the
kinetic intermediacy of EI between E·I and
E*I*, our results also permit the interesting inference that
the P1-P1
linkage remains intact in EI
. Only a minor
amount of cleaved I is formed during the course of reaction of
-chymotrypsin and either rACT or MCM-A352C-rACT (Fig. 1). Furthermore, at 40 °C, the rate of cleaved I formation was clearly much slower than the rate of fluorescence change. These observations require that when EI
dissociates during the quench/analysis
procedure, it forms E and intact I, whereas cleaved I would
have been expected if the P1-P1
linkage were cleaved in
EI
. Here it should be noted that EI
rises to a
level of ~35-40% of total I after 40 ms, so cleaved I arising from
EI
dissociation would have been easily detectable.
Interpreted according to Scheme 2, maintenance of the P1-P1
linkage
in EI
requires that partitioning into substrate and
inhibitor pathways occurs prior to acyl-enzyme formation.
The small accumulation of cleaved I may reflect formation of cleaved I in solution (via reaction 4 in Scheme 2), due to partitioning of the E·I complex in solution between inhibitor and substrate pathways, or may rather arise from partitioning of E*I* in solution into an SDS-stable complex (the major pathway) and an SDS-unstable complex as a consequence of the quench/analysis procedure. Given the evidence that E*I* may correspond to acyl-enzyme or to the final tetrahedral intermediate (35) resulting from water attack on the acyl-enzyme, an interesting possibility is that the observed cleaved I may arise from the breakdown of the final tetrahedral intermediate during quench/analysis.
A final speculation, based on the pH dependence of
kf, is that EI corresponds to a covalent
adduct between E and I, as suggested by Olson et
al. (48). More specifically, we propose the initial tetrahedral
adduct formed by serine 195 attack on the P1 carbonyl. Such an adduct
could reasonably be expected to break down to E and intact I
on quench/analysis. Experiments underway to directly measure the rate
of P1-P1
cleavage within the complex should permit direct testing of
whether the P1-P1
linkage is intact within EI
.
We thank Michael Plotnick and Norman Schechter for helpful discussions and advice. We acknowledge the excellent technical assistance in several aspects of this work of Nora Zuño, Trevor Selwood, Xuzhuo Liu, and Qiuhui Zhong.