From the Laboratoire d'Enzymologie, INSERM U392,
Université Louis Pasteur de Strasbourg, 67400 Illkirch, France,
and the § Department of Biochemistry, University of
Cambridge, Cambridge CB2 1QW, United Kingdom
Received for publication, October 2, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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Neutrophil proteinase 3 (Pr3) and elastase (NE)
may cause lung tissue destruction in emphysema and cystic fibrosis.
These serine proteinases have similar P1
specificities. We have compared their catalytic activity using
acyl-tetrapeptide-p-nitroanilides, which occupy the
S5-S'1 subsites of their substrate binding
site, and intramolecularly quenched fluorogenic heptapeptides,
which bind at S5-S'4. Most
p-nitroanilide substrates are turned over slowly by Pr3 as
compared with NE. These differences disappear with the fluorogenic
heptapeptides, some of which are hydrolyzed even faster by Pr3 than by
NE. Elongation of substrates strongly increases the catalytic
efficiency of Pr3, whereas it has little effect on NE catalysis. These
different sensitivities to S'-P' interactions show that Pr3 and NE are
not interchangeable enzymes despite their similar P1 specificity.
The azurophilic granules of polymorphonuclear
neutrophils contain three serine proteinases: elastase
(NE),1 cathepsin G, and
proteinase 3 (Pr3), which participate in lysosomal bacterial digestion
and neutrophil migration through the extracellular matrix at sites of
inflammation. These enzymes are ~30-kDa glycoproteins, which belong
to the chymotrypsin family of serine proteinases. Pr3 is the most
recently discovered, the most difficult to isolate, and hence the less
well studied proteinase of the three. It is identical to three
independently discovered proteins: (i) myeloblastin, which regulates
the growth and differentiation of leukemic cells; (ii) p29b, which has
microbicidal activity; and (iii) the target antigen of antineutrophil
cytoplasmic autoantibodies detected in patients with Wegner's
granulomatosis (2). Pr3 cleaves extracellular matrix proteins including
elastin, type IV collagen, fibronectin, laminin, and vitronectin (3,
4). It is able to produce lung emphysema in hamsters (3), and thus, in
concert with NE and cathepsin G, it may be responsible for lung tissue
destruction occurring in emphysema and cystic fibrosis.
Although many model substrates have been used to map the
active site of NE (5-8), literature on the substrate specificity and
the catalytic activity of Pr3 is poorly documented. Fuginaga et
al. (9) have shown that the two enzymes have similar substrate binding sites. This explains why both proteinases cleave the oxidized insulin A and B chains at peptide bonds involving small aliphatic amino
acid residues (4). The specificity of Pr3 for non-bulky residues has
been confirmed with a limited number of model substrates (4, 10-12).
As a rule, these substrates were turned over at a much lower rate by
Pr3 than by NE, an unexplained observation. We have undertaken the
present work to understand this puzzling finding.
Materials--
NE was isolated, and active site was
titrated as described previously (13). Pr3 came from Athens Research
Technology (Athens, GA) and was titrated with
Kinetics of Substrate Hydrolysis--
All of the kinetic
measurements were done at 25 °C and pH 7.4 (50 mM Hepes,
150 mM NaCl). The enzymatic reactions were initiated by
adding a small volume of enzyme solution to the buffered substrate solution contained in a spectrophotometer or a fluorometer cuvette. The
final concentration of dimethylformamide was 5% (v/v) throughout.
The initial rate of p-nitroanilide hydrolysis was measured
at 410 nm using variable concentrations of substrate and constant concentrations of enzyme. For the most sensitive substrates, the final
Pr3 and NE concentrations were 140 and 60 nM, respectively. For the less sensitive substrates, 10-50-fold higher enzyme
concentrations were used. The kinetic parameters
kcat and Km were calculated from non-linear least square fits to the Michaelis-Menten equation (Enzfitter software). The Pr3-catalyzed hydrolysis of
Suc-Ala4-pNA was so slow that
kcat and Km could not be
determined separately. Therefore, we recorded the full hydrolysis of
10-30 µM Suc-Ala4-pNA by 3.3 µM Pr3. The
kcat/Km ratio for this
enzyme-substrate pair was calculated from the progress curve as
outlined below for the fluorogenic substrates.
The cleavage of the fluorogenic substrates was monitored at
Site of Substrate Cleavage--
The p-nitroanilide
substrates (2 µM) dissolved in the above buffer + 5%
dimethylformamide were reacted with 0.3-15 µM enzyme in
a total volume of 1 ml. After ~20% substrate hydrolysis, the reaction was stopped with 10 µl of trifluoroacetic acid, the
medium was diluted 200-fold with buffer A (buffer + 0.1%
trifluoroacetic acid), and 40 µl of this dilution was applied to a
3.9 × 100-mm C18 Novo-pack column equilibrated with buffer A. A
linear gradient formed with buffers A and B (0.1% trifluoroacetic acid
in acetonitrile) was used to separate the reaction products. Elution
was followed at 214 nm at a flow rate of 0.8 ml·min
The fluorogenic substrates (5 µM) dissolved in the buffer + 5% dimethylformamide were reacted with 30 nM to 1.6 µM enzyme in a total volume of 1 ml. After ~50%
hydrolysis, the reaction was stopped with 30 ml of glacial acetic acid.
The digests were absorbed on C18 reverse-phase cartridges, washed with
5% acetonitrile to remove the salts, and then the peptide fragments
eluted with 60% acetonitrile. The eluant mixtures were
rotary-evaporated to dryness and taken up in 0.1 ml of 60%
acetonitrile. The fragments were identified by electrospray ionization
mass spectrometry. The fragment signals were 10-100 times that of the background.
Compared Action of Pr3 and NE on Acyl-tetrapeptide
p-Nitroanilides--
All of the substrates listed in Table
I are hydrolyzed more or less
efficiently by NE, but many of them are more resistant to Pr3 than to
NE. The proteolytic coefficient
kcat/Km may be up to 380-fold
lower for Pr3 than for NE (see substrate 1). For compound
8, the best NE substrate (5), the difference is 100-fold.
With a few exceptions, the differences in reactivity are because of
differences in kcat. Very poor NE substrates are
not hydrolyzed at all by Pr3 (see nb 10, 11,
13). Also, none of the substrates
Suc-Ala-Ala-Pro-X-pNA (X = Asp, Glu, Lys,
and ornithine) is hydrolyzed by either NE or Pr3. The poor
catalytic power of Pr3 is not the result of non-productive enzyme-substrate binding because high pressure liquid
chromatography analyses showed that the only reaction products
resulting from partial hydrolysis by Pr3 (or NE) were
acyl-tetrapeptides + p-nitroaniline, which indicates
that binding involves subsites S5 to
S'12 of
the active center.
A number of compounds are cleaved by Pr3 and NE with comparable
proteolytic coefficients. These are either poor (nb 12, 14, 15) or good NE substrates (nb
18-20). Compound 19 is the only
p-nitroanilide whose kcat for Pr3 is
significantly higher than that for NE.
Compared Action of Pr3 and NE on Fluorogenic Peptides--
We have
synthesized a number of heptapeptides to cover both S and S' subsites
of the active center of Pr3 and NE. To follow their hydrolysis using
fluorescence emission, we have included the fluorescent label Mca and
the fluorescence quencher Dpa (14) at their N and C termini,
respectively. For reasons given under "Experimental Procedures,"
kcat and Km could not be
determined separately. However, their ratio is meaningful because (i)
it represents the second-order acylation rate constant whether
acylation is rate-limiting or not and whether substrate binding is
productive or not (15), and because (ii) it is a good measure of
specificity (16).
The cleavage sites of the fluorogenic substrates were determined by
electrospray ionization mass spectrometry. The masses (Da) of the
N-terminal peptides generated by Pr3 and NE were 609.5 and 609.3 for
substrate 21 (theoretical mass of Mca-Ala-Ala-Pro-Leu = 609.7);
547.1 and 545 for substrate 22 (Mca-Ala4 = 541.5); 839.7 and 839.4 for substrate 24 (Mca-L2p-Tyr-Asp-Ala = 839.8); 867.7 and 865.5 for substrate 25 (Mca-L2p-Tyr-Asp-Val = 867.9); 880.8 and 879.5 for substrate 26 (Mca-L2p-Tyr-Asp-Ile = 881.9); and 789.3 and 788.2 for substrate 27 (Mca-L2p-Val-Glu-Ala = 789.8). The N-terminal peptide generated by the action of NE on substrate 23 had a mass of 595.6 (Mca-Ala2-Pro-Val = 596.6). The
Pr3 + substrate 23 mixture did not give a clear-cut mass profile
because of paucity of the material.
Table II shows that Pr3 hydrolyzes most fluorogenic
substrates very rapidly. It is even more
effective than NE on compounds 24-27. Table II also compares the
proteolytic coefficients of Pr3 and NE on six heptapeptidic and
tetrapeptidic substrates encompassing identical N-terminal
tetrapeptidic sequences. It can be seen that the catalytic activity of
Pr3 dramatically increases with peptide chain elongation, whereas that
of NE does not significantly vary with substrate length.
Pr3 and NE belong to the chymotrypsin-like family of serine
proteinases whose active site is composed of a substrate-binding site
responsible for specificity and of a catalytic site responsible for
substrate hydrolysis. The latter comprises a highly conserved Asp-His-Ser triad whose hydrogen bonding transforms the serine O Whereas many studies have probed the S region of the substrate binding
site of serine proteinases, literature is poorly documented on the
S'-binding region (19). Stein and Strimpler (7) have shown that the
favorable effect of S'-P' interaction on NE catalysis (18) is mainly
the result of occupancy of subsite S'1 because there are no
important binding interactions available past S'1. This is
in agreement with our data showing that elongation of the NE substrates
beyond S'1 has no significant effect on catalysis.
The present work is the first report on S'-P' interactions in Pr3. It
explains why p-nitroanilides (4, 12) and thiobenzyl esters
(10, 11) were found to be poor substrates of Pr3. It also allows the
conclusion that Pr3 and NE cannot be considered as two interchangeable
enzymes despite their common specificity for small aliphatic amino acid
residues. Indeed, the divergent effects of S'-P' binding may lead to
different specificities on biological protein substrates. In this
context, it is noteworthy that Pr3 and NE show differences in the
digestion pattern of fibronectin, laminin, and collagen (4).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-proteinase inhibitor (4). This enzyme preparation was
electrophoretically pure. The presence of NE was tested by reacting
0.14 µM Pr3 with increasing amounts of secretory
leukoprotease inhibitor (1-8 µM, final concentration), which inhibits NE but does not inhibit Pr3 (3). The buffer was the same
as that used for enzyme kinetics. After 5 min at 25 °C, 2.1 mM MeOSuc-Ala2-Pro-Val-pNA, a very
sensitive NE substrate (5), was added to the mixtures and the enzymic
rates were measured at 410 nm and 25 °C. The lack of enzyme activity
indicated that this commercial preparation of Pr3 was free of
contaminating NE. Most p-nitroanilide substrates were
purchased from Bachem (Bubendorf, Switzerland).
Suc-Ala2-Asp-Val-pNA,
Suc-Ala2-Glu-Val-pNA,
Suc-Leu-Val-Glu-Ala-pNA, Suc-Ala4-pNA,
MeOSuc-L2p-Tyr-Asp-Ala-pNA, and
MeOSuc-L2p-Tyr-Asp-Val-pNA were synthesized by Enzyme System
Products (Livermore, CA). The intramolecularly quenched
fluorogenic substrates Mca-peptide-Dpa-NH2 were synthesized
using the same methodology as described earlier (14). The compounds
were purified by reverse-phase chromatography, and their identity was
confirmed by mass spectrometry. All of the substrates were dissolved in
dimethylformamide. Their concentration was checked
spectrophotometrically using
315 nm = 14,600 M
1 cm
1 and
410
nm = 7,500 M
1 cm
1 for
p-nitroanilides and fluorescent substrates, respectively.
ex = 328 nm and
em = 393 nm (14) using
substrate concentrations below 10 µM to avoid absorptive
fluorescence quenching of the hydrolysis product. The absorptive
quenching also precluded the separate measurement of
kcat and Km. Therefore, the
hydrolysis of the fluorogenic substrates was followed to completion. At
low substrate concentrations ([S]o < Km), the Michaelis-Menten equation simplifies to
v = kcat/Km × [E]o × [S]o so that the release of
product P with time is a first-order reaction described by
d[P]/dt = kobs
[S] where kobs = kcat/Km × [E]o. The pseudo first-order rate constant
kobs was calculated by non-linear regression
analysis of progress curves recorded using 0.5-5 µM substrate concentrations and 0.1-1 µM enzyme
concentrations. The progress curves were all first-order, and
kobs did not significantly change with
[S]o. This indicates that [S]o was indeed
lower than Km.
1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Kinetics of the hydrolysis of acyl-tetrapeptide p-nitroanilides by Pr3
and NE at pH 7.4 and 25 °C
Compared catalytic activity of Pr3 and NE on intramolecularly quenched
fluorogenic substrates at pH 7.4 and 25 °C
20%.
The table also compares fluorogenic (Fluo) and
p-nitroanilide substrates (from Table I) encompassing
identical N-terminal tetrapeptidic sequences. Fluo/pNA
means kcat/Km of fluorogenic
substrate/kcat/Km of
pNA substrates. The arrow indicates the cleavage points.
n.h. = no hydrolysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
into a powerful nucleophile that attacks the scissile peptide bond of
the substrate. The efficacy of catalysis is due in part to a precise
binding of substrate with resultant proper orientation of the scissile
bond with respect to O
of the catalytic serine (17). Fuginaga
et al. (9) have shown that Pr3 and NE have similar
substrate-binding sites that account for their preference for small
aliphatic residues at P1 (4). However, the use of some
model substrates and inhibitors indicated that Pr3 is a much poorer
catalyst than NE (4, 10-12). We thought this might be because of the
fact that Pr3 (but not NE) binds these compounds in a predominantly
non-productive way. Therefore, we used a series of
acyl-tetrapeptide-p-nitroanilides, which all bind
productively at subsites S5 to S'1. This
series included substrates with P2 = Asp or Glu
to explore the possibility of electrostatic
S2-P2 interactions (9). Because the catalytic
differences persisted for most of these compounds, we hypothesized that
the occupancy of subsites S5 to S'1 of Pr3 may
not be sufficient to align the substrate in a position favorable for
efficient catalysis, whereas it may be sufficient in the case of NE.
The use of fluorogenic heptapeptides confirmed our hypothesis that
elongation of the substrates strongly increased the catalytic
efficiency of Pr3, whereas it had a much less effect on NE catalysis.
Thus, the poor catalytic activity of Pr3 on tetrapeptidic substrates is
no longer observed with heptapeptidic substrates, some of which are
turned over even more rapidly by Pr3 than by NE. It is interesting to note that the rate-enhancing effect attributed to full occupancy of the
S' region of Pr3 is more pronounced for poor p-nitroanilide substrates (nb 1, 7, 11 with P4 = Ala) than for
good ones (nb 19, 20 with P4 = L2p). This
finding suggests that S4-P4 and S'-P'
interactions play a complementary role in binding substrates nb
19 and 20 in a position favorable for catalysis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Christian Boudier for assistance and the French cystic fibrosis association, Vaincre la Mucoviscidose, for financial support.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed: INSERM U 392, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France. Tel.: 33-3-90-24-41-82; Fax: 33-3-90-24-43-08; E-mail: jgbieth@aspirine.u-strasbg.fr.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210074200
2 The S and S' subsites of the substrate binding site and the corresponding P and P' residues of the substrate are labeled according to the nomenclature of Schechter and Berger (1).
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ABBREVIATIONS |
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The abbreviations used are: NE, human neutrophil elastase; Pr3, human neutrophil proteinase 3; Suc, succinyl; MeOSuc, methoxysuccinyl; Ac, acetyl; Boc, tert-butyloxycarbonyl; pNA, p-nitroanilide; L2p, lysyl-(2-picolinoyl); Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; nb, number.
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