(Received for publication, December 23, 1996, and in revised form, March 3, 1997)
From the Center for Molecular Biology of Oral
Diseases and ¶ Department of Oral Medicine and Diagnostic
Sciences, University of Illinois, Chicago, Illinois 60612-7213 and the
Department of Veterinary Medical Chemistry, Swedish University
of Agricultural Sciences, Uppsala Biomedical Center,
S-751 23 Uppsala, Sweden
Protein proteinase
inhibitors of the serpin family were recently reported to form
SDS-stable complexes with inactive serine proteinases modified at the
catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the
complexes formed with the active enzymes (Christensen, S., Valnickova,
Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P.,
and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports
that similar active site modifications of serine proteinases block the
ability of serpins to form SDS-stable complexes prompted us to
investigate the mechanism of complex formation between serpins and
DCI-inactivated enzymes. Both neutrophil elastase and -trypsin
inactivated by DCI appeared to form SDS-stable complexes with the
serpin,
1-proteinase inhibitor (
1PI), as reported previously. However, several observations suggested that such
complex formation resulted from a reaction not with the DCI enzyme but
rather with active enzyme regenerated from the DCI enzyme by a
rate-limiting hydrolysis reaction. Thus (i) complex formation was
blocked by active site-directed peptide chloromethyl ketone
inhibitors; (ii) the kinetics of complex formation indicated that
the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on
inhibitor concentration; and (iii) complex formation was
accompanied by stoichiometric release of a peptide having the
sequence SIPPE corresponding to cleavage at the
1PI
reactive center P1-P1
bond. Quantitation of kinetic constants for DCI
and
1PI inactivation of human neutrophil elastase and
trypsin and for reactivation of the DCI enzymes showed that the
observed complex formation could be fully accounted for by
1PI preferentially reacting with active enzyme
regenerated from DCI enzyme during the reaction. These results support
previous findings of the critical importance of the proteinase
catalytic serine in the formation of SDS-stable serpin-proteinase
complexes and are in accord with an inhibitory mechanism in which the
proteinase is trapped at the acyl intermediate stage of proteolysis of
the serpin as a substrate.
Serpins comprise a large superfamily of proteins, many of which regulate the activity of serine and, in some cases, also cysteine proteinases in numerous biological processes by functioning as inhibitors of these proteinases (1, 2). Such serpins inhibit their target proteinases by a mechanism that bears similarities to that of other nonserpin families of protein proteinase inhibitors (3, 4). Serpin and nonserpin inhibitors thus both possess an exposed reactive center loop that binds to the active site of their target proteinases in the manner of a substrate. Additionally, both types of inhibitors trap proteinases in stable complexes at an intermediate stage of proteolysis of the inhibitor reactive center loop as a substrate. However, the nature of the trapped complexes distinguishes serpin from nonserpin inhibitors. Serpin-proteinase complexes are thus stable in denaturants such as SDS and guanidinium chloride (5-8), whereas nonserpin inhibitor-proteinase complexes dissociate under such conditions (9). Serpins must further undergo a major conformational change to trap their target proteinases in stable complexes (10-15), whereas nonserpin inhibitors require minimal conformational adjustments to bind their target enzymes (3, 4). Such differences in behavior have suggested that serpins have evolved a novel mechanism for inhibiting proteinases that greatly differs from the mechanism used by nonserpin inhibitors.
While there are no available structures of serpin-proteinase complexes, the SDS stability of these complexes has suggested that serpins may trap proteinases at the tetrahedral or acyl intermediate stage of proteolysis of the serpin as a substrate (2). Early studies favored trapping of the proteinase at the acyl intermediate stage of proteolysis, based on the observation that the serpin reactive center loop was cleaved in the stable complex with proteinase (8, 16-18). However, NMR evidence later suggested that stabilization of the serpin-proteinase complex at the acyl intermediate stage occurred only under denaturing conditions and that under native conditions, the complex was stabilized at the tetrahedral intermediate stage (19). Yet more recent findings have questioned the NMR data by showing that, even under nondenaturing conditions, the reactive center loop is cleaved concomitant with formation of the serpin-proteinase complex, thus supporting an arrest of the serpin-proteinase reaction at the acyl intermediate stage (20-22).
Further controversy has ensued from another recent report that has suggested that the SDS stability of serpin-proteinase complexes does not result from a covalent linkage of the inhibitor to the enzyme active site as would exist in an acyl or tetrahedral intermediate, but rather is due to a covalent cross-linking of the inhibitor with proteinase outside of the enzyme active site (23). The evidence supporting this conclusion included the observations that (i) proteinases blocked at the active site by the inactivating reagent, 3,4-dichloroisocoumarin (DCI),1 appeared still capable of forming SDS-stable complexes with serpins and (ii) no release of the carboxyl-terminal region of the serpin from the complex with proteinase could be demonstrated on denaturation, implying that cleavage of the reactive center loop of the serpin had not occurred. These observations contrast with several earlier studies that showed that the blocking of the proteinase active site with reagents such as diisopropyl fluorophosphate prevented complex formation with serpin inhibitors (5, 7, 24). Even allowing that the DCI reagent is small enough to permit binding of the serpin at the enzyme active site, the results of this study still disagree with other findings with active site-modified serine proteinases, in which minimal modifications of the active site serine to alanine or to dehydroalanine or of the active site histidine to methylhistidine were made. Such catalytically inactive modified enzymes were thus found to be capable of forming only noncovalent complexes with serpins that were dissociable in SDS (7, 9, 25-27). Further, these complexes were of much lower affinity than the SDS-stable complexes formed with active proteinases, consistent with a covalent interaction involving the proteinase catalytic serine residue stabilizing the serpin-proteinase complexes (9).
Because the findings of Christensen et al. (23) are diametrically opposed to previous findings regarding the nature of the stable serpin-proteinase complex, we sought to determine the basis for the discrepancies between these studies by investigating the mechanism by which DCI proteinases form SDS-stable complexes with serpins. While the present study confirms that SDS-stable complexes are formed in the reaction of DCI proteinases with serpins, our reinvestigation of this phenomenon clearly demonstrates that such complexes do not arise from the DCI-inactivated enzyme but rather are formed from active enzyme regenerated from the DCI enzyme. Moreover, evidence is presented that the serpin reactive bond is cleaved in the SDS-stable complexes formed with the DCI enzyme, consistent with the complexes arising from active enzyme and involving a trapping of the enzyme at the acyl intermediate stage of proteolysis.
Bovine trypsin (Type XIII, Sigma) was purified by
soybean trypsin inhibitor-agarose affinity chromatography to isolate
the single chain -form of the enzyme (28). Autolysis of the enzyme was prevented by storage in 1 mM HCl, 10 mM
CaCl2. Human neutrophil elastase (HNE) and human
1-proteinase inhibitor (
1PI) were
purchased from Athens Research and Technology Inc. (Athens, GA).
Alternatively,
1PI was purified from outdated plasma as
described (9).
-Trypsin concentrations were determined from initial
rates of hydrolysis of the chromogenic substrate, S-2222 (Pharmacia
Hepar, Franklin, OH), by the enzyme using the turnover number measured
with active site titrated enzyme (29). Comparison with the
concentration measured from the 280-nm absorbance using an absorption
coefficient of 36,800 M
1 cm
1
(30) indicated the enzyme was
90% active. The concentration of
1PI was determined from the 280-nm absorbance based on
an absorption coefficient of 25,400 M
1
cm
1 (31). The inhibitor was fully active based on the 1:1
inhibition stoichiometry (within 10%) measured by titration of
-trypsin with
1PI (9). HNE concentrations were
determined by titration with
1PI assuming an equimolar
inhibition stoichiometry.
Trypsin experiments were conducted in 0.1 M Hepes, 0.1% polyethylene glycol 8000, pH 8.0, and HNE experiments were conducted in the same buffer plus 0.5 M NaCl. Temperatures were either 25 or 37 °C as indicated.
Kinetics of DCI HydrolysisDCI (Sigma) was dissolved in
dimethyl sulfoxide (Me2SO) that had been dried by storage
over molecular sieves (Type 4A, Fisher). Hydrolysis reactions were
initiated by diluting 0.1 ml of DCI into 0.9 ml of either trypsin or
HNE buffers to give final DCI concentrations of 125-250
µM and 10% Me2SO. Hydrolysis was
continuously monitored from the decrease in absorbance at 325 nm for
several half-lives (32). First-order rate constants were obtained by nonlinear regression fitting of reaction curves by an exponential function with a floating end point. Indistinguishable rate constants were obtained at the two DCI concentrations. DCI concentrations were
calculated from the initial 325-nm absorbance using an absorption coefficient of 3330 M1 cm
1
(32).
The kinetics of enzyme inhibition were measured under pseudo first-order conditions either by discontinuous or continuous assay methods (33). For the discontinuous assay method, reactions contained 10 nM enzyme and either 125-250 µM DCI, 100-200 nM Phe-Phe-Arg chloromethyl ketone (Calbiochem), or 5 µM methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (Bachem, King of Prussia, PA) in 0.1 ml. Identical reaction samples were quenched at various times with 0.9 ml of chromogenic substrate, and residual enzyme activity was measured from the initial rate of substrate hydrolysis at 405 nm. The substrates were 200 µM methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Calbiochem) for HNE and 200 µM S-2222 for trypsin. For reactions of 4-10 µM trypsin with 250 µM DCI at 25 °C, samples of the reaction mixture were first quenched at different times into 4 mM HCl, 10 mM CaCl2 (40-fold dilution) and then further diluted 100-fold into substrate for activity determination. Pseudo first-order rate constants (kobs) were determined by nonlinear regression fitting of the loss of enzyme activity to an exponential decay function. Second-order rate constants were obtained by dividing kobs by the inhibitor concentration. For the continuous assay, DCI and then HNE were added in rapid succession to chromogenic substrate to give final concentrations of 20 nM HNE, 2.5-10 µM DCI, and 320 µM substrate. The exponential decrease in substrate hydrolysis rate was monitored continuously for up to 5 min to an end point rate (<2% of the initial rate) with <5% consumption of substrate. Progress curves were fit by nonlinear regression to an exponential plus linear term (33, 34) to obtain kobs. kobs was corrected for substrate competition by multiplying by the factor, 1 + [S]0/Km, where [S]0 is the substrate concentration. Km was measured to be 46 ± 9 µM at 25 °C and 52 ± 4 µM at 37 °C (±2 S.E.). Second-order rate constants were obtained by dividing the corrected kobs by the inhibitor concentration. The final concentration of Me2SO was 10% in all DCI enzyme reactions. No correction for hydrolysis of DCI was made in any of these reactions, since the extent of such hydrolysis was found to be negligible over the time course of the reactions studied.
Kinetics ofThe kinetics of inhibition of proteinases by
1PI were measured under pseudo first-order conditions
either by discontinuous (trypsin) or continuous (HNE) assays. Trypsin
reactions contained 2.5 nM enzyme and 25 or 50 nM
1PI in 0.1 ml. At different times, reactions were quenched by adding 0.9 ml of 200 µM S-2222
substrate, and residual enzyme activity was measured from the initial
substrate hydrolysis rate at 405 nm. kobs was
determined by nonlinear regression fits to an exponential decay
function. HNE reactions contained 1.25-5 nM
1PI, 0.25-1 nM HNE, and 330 µM substrate and were initiated with enzyme. The
exponential decrease in substrate hydrolysis rate was monitored
continuously until an end point rate was reached (<1% of the initial
rate) during which <1% substrate was consumed. Progress curves were
fit by an exponential plus linear function to obtain
kobs, and second-order rate constants were
obtained from corrected values of kobs, as
described for DCI HNE reactions.
DCI enzyme was prepared by incubating 250 µM DCI with 6 µM enzyme for 15 min at
25 °C (final Me2SO concentration, 10%). After diluting
the inactivated enzyme 20-fold, excess DCI was removed by dialysis at
4 °C for 3 h against 100 volumes of reaction buffer containing
4-5% Me2SO (to mimic the conditions for
1PI complex formation in SDS gel electrophoresis
experiments) with buffer changes after each hour. A control enzyme
sample was treated similarly except that no DCI was added. A 50-µl
aliquot containing 47-140 nM DCI HNE or 5-16
nM DCI trypsin was then added to 0.95 ml of 200 µM substrate in buffer containing 4-5%
Me2SO, and the accelerating rate of substrate hydrolysis
was monitored continuously at 405 nm for 15 min at 37 °C (
5%
substrate hydrolysis). Data were fit by nonlinear regression by the
parabolic equation (33),
![]() |
Reactions of 1PI with active and
DCI-inactivated enzymes followed the protocol of Christensen et
al. (23). DCI enzymes were prepared by incubating enzyme with 250 µM DCI for 15 min at 25 °C (final Me2SO
concentration, 10%). An equal volume of
1PI or
1PI plus Phe-Phe-Arg chloromethyl ketone or
methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone at 37 °C was
then added in a single aliquot, and the incubation was continued at
37 °C for 30 min. Reactions were then quenched by first adding
either 500 µM Phe-Phe-Arg chloromethyl ketone (trypsin)
or 250 µM DCI (HNE) to avoid nonspecific proteolysis during denaturation followed by addition of SDS and then boiling for 3 min before electrophoresis on 10% gels according to Laemmli (35). For
experiments designed to detect a carboxyl-terminal peptide formed
concomitant with the
1PI-proteinase complex, a Tricine-step gel system was used, in which the lower half of the gel
was 16.5% acrylamide and the upper half was 10% acrylamide (36).
Bands were stained with Coomassie Brilliant Blue R-250 and quantified
by scanning the gels in an UltroScan XL laser densitometer (Pharmacia-LKB Biotechnology, Uppsala, Sweden). For the quantitation of
serpin-proteinase complex formation in
1PI reactions
with DCI enzymes, the amount of complex formed in the reaction with active enzyme was taken as 100%. A series of dilutions of this maximum
level of complex indicated a linear relationship between the amount of
complex and the integrated band intensity. For Western blotting
detection of
1PI·HNE complexes formed at lower serpin and proteinase concentrations, proteins electrophoresed on 10% SDS
gels were transferred to nitrocellulose membranes (37), and inhibitor
bands were then detected by incubating with sheep anti-
1PI Ig followed by donkey anti-sheep IgG conjugated
with alkaline phosphatase (Binding Site, Inc., San Diego, CA) and then 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma).
After SDS-Tricine step polyacrylamide gel electrophoresis to separate the carboxyl-terminal peptide as described above, the unstained gel was electroblotted to a polyvinylidene difluoride membrane (Qiabrane PVDF, Qiagen, Chatsworth, CA). The membrane was stained with Coomassie Brilliant Blue R-250, the appropriate band was excised, and the peptide was sequenced directly in an Applied Biosystems (Foster City, CA) 470A gas phase sequencer connected on line to a 120A phenylthiohydantoin analyzer.
In the reaction of DCI trypsin with 1PI, an
SDS-stable complex was formed that was indistinguishable from the
complex formed with active trypsin, in agreement with the report of
Christensen et al. (23) (Fig. 1). However, as
also observed in the previous report, the amount of complex formed when
DCI trypsin was reacted with equimolar
1PI for 30 min at
37 °C was considerably less than that formed with active trypsin
under identical conditions (Fig. 1, lanes 4 and
7). Doubling the concentration of DCI enzyme in the reaction
doubled the amount of complex, whereas varying the
1PI
concentration from 0.5 to 2 times the molar concentration of DCI enzyme
produced the same amount of complex as that formed at equimolar
concentrations (Fig. 1, lanes 7-9). The extent of complex
formation at equimolar DCI enzyme and inhibitor also progressively increased with increasing time of incubation (Fig. 2,
lanes 4-6). Integrating the intensity of the complex bands
in a separate experiment revealed that 14, 25, and 39% of the complex
produced with active enzyme was formed in the reaction with the DCI
enzyme after 30, 60, and 120 min, respectively. Together, these
observations indicated that the formation of a stable complex from DCI
enzyme and
1PI was limited by a reaction that was first
order in DCI enzyme concentration but zero order in inhibitor
concentration. This contrasts with the
1PI reaction with
active trypsin, which is first order in both enzyme and inhibitor
concentrations, i.e. second order overall.
To assess whether the SDS-stable complexes formed with
1PI and DCI trypsin involved a covalent interaction
between the enzyme and inhibitor outside the enzyme active site rather
than within the active site, we tested the effect of an efficient
active site-directed inhibitor, D-Phe-Phe-Arg chloromethyl
ketone, on SDS-stable complex formation. Addition of chloromethyl
ketone together with
1PI resulted in a nearly complete
blocking of the reaction of
1PI with both active trypsin
and inactive DCI trypsin (Fig. 1, lanes 5 and
10). Such results suggested that the complex formed in the reaction of DCI enzyme with
1PI might be arising from
active enzyme generated during the reaction by hydrolysis of the DCI inactivating group rather than from the DCI enzyme itself.
To investigate this possibility, the kinetics of trypsin inactivation
by DCI and reactivation of DCI trypsin were analyzed under the
conditions used for observing SDS-stable complex formation between 1PI and DCI trypsin in the report of
Christensen et al. (23). The enzyme (4-10
µM) was inactivated by 250 µM DCI at 25 °C in a first-order process (t1/2 ~30 s),
with less than 1% residual activity remaining after the 15-min
incubation previously used to prepare the DCI enzyme. The second-order
rate constant determined from inactivation progress curves over a range
of inhibitor concentrations and under pseudo first-order conditions was
about 2-fold lower than the value originally reported for this reaction
by Harper et al. (32) at lower pH and higher ionic strength
(Table I). Although trypsin appeared to be essentially
all inactivated by DCI, DCI enzymes are known to slowly regenerate
active enzyme by hydrolysis of the active site blocking reagent (32).
Such regeneration of trypsin from DCI trypsin was measured by diluting the DCI enzyme into a reporter chromogenic substrate after dialysis to
remove excess DCI and monitoring the acceleration of the substrate hydrolysis rate. A first-order rate constant of 8.1 ± 0.6 × 10
5 s
1 was determined for release of active
enzyme under the conditions used for complex formation with
1PI, i.e. 37 °C. Based on this rate
constant, the amount of reactivated trypsin formed was predicted to be
14 ± 1% during the 30-min time interval allowed for reaction of
DCI trypsin with
1PI in Figs. 1 and 2, similar to the
13-15% complex observed by integrating the intensity of the complex
bands. This observation was in keeping with the expectation that any trypsin regenerated from DCI trypsin would exclusively react with
1PI at the concentrations of serpin and DCI present
during the reaction, given the ~5000-fold faster second-order rate
constant measured for inhibition of trypsin by
1PI than
by DCI and the depletion of DCI by hydrolysis during the formation of
the DCI enzyme (Table I and Scheme 1). The ability to
block the reaction of regenerated trypsin with
1PI by
Phe-Phe-Arg chloromethyl ketone was also consistent with the relative
inhibition rate constants and concentrations of serpin and chloromethyl
ketone employed in the reaction (Table I).
|
The above observations indicated that the amount of SDS-stable complex
formed in the reaction of 1PI with DCI trypsin could be
fully accounted for by the amount of active trypsin regenerated from
DCI trypsin by a rate-limiting hydrolysis of the inactivated enzyme
(Scheme 1). Such an interpretation was consistent with the first-order
dependence of complex formation on the DCI enzyme concentration, the
independence on the
1PI concentration, and the
dependence of the amount of complex formed on the time of incubation of
DCI trypsin with
1PI. In keeping with this conclusion, a
~5-kDa peptide appeared concomitant with the formation of the SDS-stable complex in the reactions of
1PI with both DCI
trypsin and trypsin (Fig. 2, lanes 3-6). The amount of
peptide generated was stoichiometric with the amount of complex, based
on the similar ratios of the integrated intensities of peptide to
complex bands for active and inactive enzyme reactions. Amino-terminal
sequencing of the peptide produced in the DCI trypsin reaction gave the
sequence SIPPE corresponding to cleavage of
1PI in the
reactive center Met358-Ser359 P1-P1
bond in
the stable complex. Such cleavage is known to accompany the
reaction of
1PI with active proteinases (16, 20, 21) and
indicates that the SDS-stable complexes formed between
1PI and DCI trypsin or trypsin both result from a
reaction of the serpin with active trypsin.
Our finding of the stoichiometric release of a carboxyl-terminal
peptide from the enzyme-serpin complex resulting from cleavage at the
serpin reactive bond argues against suggestions by Christensen et
al. (23) that the carboxyl terminus of the serpin in SDS-stable 1PI-enzyme complexes is intact. The failure to observe
this peptide in the previous work may have been due to the loss or
reduced visibility of the ~5-kDa peptide in the SDS gradient gel
system employed. By increasing the amount of protein loaded on the gel and calibrating the gel with standard peptides, the ~5-kDa peptide was clearly visible in our gel system. The cleavage and release of
homologous carboxyl-terminal peptides from other SDS-stable serpin-proteinase complexes have been convincingly shown in numerous other studies (8, 16, 18, 20-22, 38).
Similar experiments comparing the reactions of 1PI with
HNE and DCI HNE showed that the DCI enzyme formed 18 ± 2% of the SDS-stable complex generated in the reaction of equimolar
1PI with active enzyme (Fig. 3, top
panel, lanes 3 and 6). The DCI HNE reaction
showed a similar first-order dependence of stable complex formation on
the concentration of DCI enzyme, independence of inhibitor
concentration, and dependence on the time of reaction, as the DCI
trypsin reaction (Fig. 3, top panel, lanes 6-8).
Measurement of the rate constants for the inactivation of HNE by DCI
and
1PI and for the regeneration of HNE from DCI HNE
(Table I) predicted that 22 ± 2% of the stable complex should
result from the regeneration of active HNE during the 30-min reaction
with
1PI, in good agreement with the amount observed.
Addition of an HNE-specific tetrapeptide chloromethyl ketone reagent (2 mM methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone)
together with
1PI was not as effective in blocking
complex formation with the DCI enzyme in this case (Fig. 3, top
panel, lanes 4 and 9). This could be
explained by the chloromethyl ketone not being an effective competitor
of
1PI at the concentrations employed, based on measured
rate constants for these two inhibitors (Table I). Consistent with this
explanation, lowering the inhibitor and DCI enzyme concentrations in
the reaction by 5-fold but maintaining the chloromethyl ketone
concentration to reduce the competitive advantage of
1PI
resulted in the ability of the chloromethyl ketone to significantly
block SDS-stable complex formation, as revealed by Western blotting
(Fig. 3, bottom panel). The SDS-stable complex formed in the
reaction of
1PI with DCI HNE was also accompanied by the
liberation of the same ~5-kDa peptide observed in the reaction of the
serpin with DCI trypsin, suggesting that the
1PI
reactive center was similarly cleaved in this complex (Fig. 2,
lanes 7 and 8). Together, these observations
provide additional evidence that the SDS-stable serpin-enzyme complexes
formed in the reactions of
1PI with either DCI HNE or
DCI trypsin arise from a reaction of the serpin with active enzyme
generated from the inactive enzyme by spontaneous hydrolysis (Scheme
1). Complex formation between DCI enzymes and serpins reported in
another study may similarly have resulted from regeneration of active
enzyme (39).
The present findings argue strongly against the conclusion made by Christensen et al. (23) that serpins can form SDS-stable complexes with proteinases that lack a functional catalytic apparatus. Several early studies clearly showed that blocking of the active site of proteinases by bulky reagents such as diisopropyl fluorophosphate prevents complex formation with serpin inhibitors (5, 7, 24). Subsequent studies in which inactivating modifications of the catalytic serine or histidine residues were made without introducing bulky groups into the active site, i.e. by converting the serine to dehydroalanine or alanine or by methylating the histidine, were also found to prevent SDS-stable complex formation with serpins. However, such active site-modified enzymes were capable of forming noncovalent complexes with serpin inhibitors (7, 9, 25-27). Significantly, the affinity of these noncovalent complexes was found to be at least 4-6 orders of magnitude weaker than that of the covalent complexes formed between the same serpins and the active proteinase (9). The observation that methylation of the active site histidine resulted in much weaker noncovalent complexes than the serine modifications (26) also implies that the DCI-inactivating group is most likely too large to allow DCI-inactivated enzymes to make any significant noncovalent interaction with serpins.
In summary, the present studies together with past findings demonstrate the critical importance of the active site catalytic serine residue of the proteinase in the formation of high affinity covalent complexes with serpins. This characteristic feature of the interactions of serpin family inhibitors with their target proteinases sets them apart from the nonserpin protein proteinase inhibitors, which are known to form tight noncovalent complexes with both active proteinases and catalytically inactive anhydroproteinases (40, 41). Our observations thus support the growing body of evidence that the inhibitory mechanism of serpins differs fundamentally from that of the nonserpin protein proteinase inhibitors. Indeed, a consensus view that has been developing is that serpins behave as suicide inhibitors, i.e. they are activated by proteinase-mediated cleavage of the serpin as a normal substrate to trap their target proteinases through a major serpin conformational change (2).