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J. Biol. Chem., Vol. 275, Issue 25, 18657-18663, June 23, 2000
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From the
Received for publication, January 12, 2000, and in revised form, March 10, 2000
The enzymatic processing of bovine collagen I by
neutrophil collagenase (MMP-8) has been monitored at 37 °C,
envisaging the occurrence of multiple intermediate steps, following the
initial cleavage, which leads to the formation of 1/4 and
3/4 fragments. Further, the first cleavage event has been
investigated at 37 °C as a function of pH, and catalytic parameters
have been obtained through a global analysis of steady-state kinetic
data, such as to get an overall consistent picture of
kcat/Km,
kcat, and Km. These data
have been compared with those obtained from the catalysis by MMP-8 of
two synthetic fluorogenic substrates under the same experimental
conditions. The overall behavior can be accounted for by the existence
of five protonating groups, which vary to a different extent their
pKa values for the three substrates investigated.
The main observation concerns the fact the two of these residues, which
play a relevant role in the enzymatic activity of MMP-8, are relatively
far from the primary recognition site, and they are coming into action
only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors
of the enzymatic action of MMP-8, which must take into account, and
also of these relevant interactions occurring only with natural substrates.
Matrix metalloproteinases
(MMP)1 are a class of
proteolytic enzymes characterized by the presence of a Zn2+
atom in the active site, which are able to process enzymatically several components of the extracellular matrix, such as interstitial and basement membranes, collagen, fibronectin, and laminin. Because of
their crucial role in the extracellular matrix degradation, they are
implicated in the tissue remodeling processes associated to the growth
and development and in several pathological conditions, such as
rheumatoid arthritis and tumor invasion (1-3). Collagenases, such as
fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and
collagenase-3 (MMP-13), are a class of MMPs that specifically cleave
several types of native triple-helical collagen (4-6), and in
particular collagen I, at a specific peptide bond between
Gly775 and the residue in position 776 (which can be either
Leu or Ile) (4, 7), leading to the formation of 1/4 and
3/4 fragments. This event is a crucial one because of the fairly
slow rate of collagen turnover within the cartilage (8), and it is
closely related to the occurrence of important physiological and
pathological events (9-11).
Collagen I is one of the major constituents of the extracellular matrix
for several tissues, such as skin, tendon, blood vessels, cartilage,
bones, and basal laminas (12), and its triple-helical structural
arrangement has been the object of several recent studies both on the
native molecule (Ref. 13 and references quoted therein) and on model
peptides (14, 15). The investigation on the action of MMPs on collagen
has been carried out for a long time, and preliminary measurements have
been carried out on the activation energy of the catalysis employing
porcine collagenases and gelatinases (16). In addition, previous
experiments have been reported on the cleavage of native and
site-directed mutants of murine collagen by MMP-1, outlining crucial
aspects of the recognition mechanism (17). Very recently, proteolysis
of single collagen I molecules has been followed by atomic force
microscopy (18), confirming that the process follows a simple
Michaelis-Menten mechanism and outlining that the results on bulk
solution are compatible with those observed on single collagen molecules.
Therefore, the knowledge of the mechanism of collagen cleavage by
collagenases indeed is very important for the comprehension of the
physiological and pathological processes affecting the extracellular
matrix and in perspective for a more effective pharmacological approach
to control and eventually inhibit the action "in vivo" of these MMPs. A previous modeling of different cleavage sites of
collagen molecules pointed out the possibility that the interaction surface between collagen and collagenases was fairly extended (19). In
the present study we have characterized the proton-linked behavior of
the proteolytic process leading to the formation of the 1/4 and
3/4 fragments of bovine collagen I by neutrophil collagenase MMP-8 by comparing it to the behavior of the same enzyme with two
fluorogenic synthetic substrates to obtain some initial information on
the different recognition and cleavage mechanisms operating with small
synthetic as well as large macromolecular substrates.
Recombinant purified neutrophil collagenase MMP-8 was kindly
obtained from Dr. G. Murphy (Strangeways Res. Laboratory, Cambridge, United Kingdom). Purity of MMP-8 was measured by SDS-polyacrylamide gel
electrophoresis according to Laemmli (20). After the gels were run,
they were stained using a silver staining kit (Bio-Rad). Zymography was
performed as follows: 2 µl of purified MMP-8 was mixed with a 5-fold
excess of sample buffer (0.25 M Tris, 0.8% SDS, 10%
glycerol, and 0.05% bromphenol blue) and run on 12%
SDS-polyacrylamide gels containing either 1 mg/ml of gelatin or
collagen type I, as described previously (21). After electrophoresis,
SDS was removed from gels by washing twice for 15 min in 2% Triton
X-100. The gels were then incubated at 37 °C for 18 h in the
incubation buffer (50 mM Tris/HCl buffer, pH 7.6, 0.15 M NaCl, 10 mM CaCl2, 2% Triton
X-100), stained with 0.5% Coomassie Blue, and destained in 10% acetic
acid and 40% methanol until pale proteinase bands were clearly
visible. The proteinase band was further characterized by adding 20 mM EDTA or 0.3 mM 1,10-phenanthroline (MMP
inhibitors) or 1 mM phenylmethylsulfonyl fluoride (serine
proteinase inhibitor) in the incubation buffer. Protein markers (Sigma)
were used as the molecular weight standard.
Recombinant purified MMP-8 proenzyme was activated by incubating 100 µl of a 0.1 µg/ml procollagenase solution with
p-aminophenyl merculric acetate (Sigma) at 37 °C for
2 h; this treatment shifts the equilibrium of the conformation
toward the open, autocatalytically activated form involving the
cleavage of the region between residues 71 and 81. Because BB-94 (known
also as Batimastat, a peptidomimetic MMPs inhibitor, kindly provided by
British Biotech Pharmaceutical, Cowley, Oxford, UK) fully inhibits
stoichiometrically MMPs, we employed it to titrate the active amount of
the enzyme.
Bovine collagen I was dissolved in acidified water at room temperature
for 2 days. Afterward, the suspension was centrifuged for 1 h at
10,000 × g, and the supernatant containing
dissolved collagen was used. The amount of collagen was quantified as
described by Bradford (22).
Fragmentation Experiments--
Activated MMP-8 (0.01 nM final concentration) in 50 mM Tris/HCl, 0.1 M NaCl, 10 mM CaCl2 has been added
to different amounts of bovine collagen (between 0.3 and 6 µM, spanning the range of Km) diluted
in the same buffer.
Different experiments were performed by using different pH values.
Mixtures were kept at 37 °C, and a small amount was harvested at
different time intervals and frozen at Circular Dichroism Experiments--
For spectroscopic
observations bovine collagen I was entrapped in agarose gel by mixing
the solubilized protein to a 0.6% low melting point agarose solution
at pH 3.0. The temperature of the agarose solution when collagen was
added was 37 °C. After rapid stirring the mixture was poured on a
simple gel casting (Mini-protean II, Bio-Rad) and immediately cooled to
obtain the gel. Final concentrations were 0.5% for agarose and 1 µM for collagen. The thickness of the homogeneous gel was
1 mm. Shares of suitable size were cut off from the homogeneous gel and
kept overnight in buffer solution to reach the desired pH without the
formation of aggregates and then used for circular dichroism measurements.
Circular dichroism (CD) spectra in the far UV region (240-190
nm) were carried out on a Jasco J710 spectropolarimeter. Each spectrum
was the average of four measurements. As a starting control, CD
measurements were performed on samples of gel-entrapped collagen at pH
3.0 as well as on solubilized collagen at the same pH and concentration
conditions. Control CD measurements have been also performed by using
agarose gel. Data obtained were considered as background.
The effect of proteolytic fragmentation of bovine collagen I by MMP-8
has been also followed by CD spectra, diffusing MMP-8 (to a final
concentration of 0.01 nM) into the slices of agarose gel
containing bovine collagen I (see Fig. 1B).
Enzymatic Assay--
Enzyme activity was measured employing two
fluorogenic substrates, namely
MCA-Pro-cyclohexylalanine-Gly Data Analysis--
Values of observed
kcat/Km,
kcat, and Km for MMP-8 at any
given pH and the pH dependence over the range investigated
(i.e. between 4.8 and 10.5) were calculated simultaneously through a global analysis of the whole data set, employing two formalisms (i.e. linear Lineweaver-Burk and sigmoidal
Michaelis) for determining the observed parameter at a given pH value.
Fitting of catalytic parameters was constrained to an internally full consistent picture, such that at any protonation level values of all
three parameters (i.e.
kcat/Km,
kcat, and Km) must be closely
related. Thus, the fitting procedure forced the system to be described
by n protonation states with n values of kcat and n values of
Km, which must combine then to give n corresponding
values of kcat/Km. The
fitting of the pH dependence is internally constrained to obey a scheme
with n values of pKU and
pKL, which are the same for all three catalytic parameters. Furthermore, because pKU values are
expected to be independent on substrate, in the fitting procedure we
have forced these values to be the same for all three substrates
(i.e. MCA-1, MCA-2, and bovine collagen I). A general
description of this system may be referred to in Scheme I
, where ES (as well as ESHx,
with × = 1, 2,..n) simply refers to the species undergoing the
rate-limiting step and xKm (with × = 0, 1,..n) refers to all preequilibrium events leading to the species
that undertake the rate-limiting step.
A comprehensive comparative global analysis has been carried out on the
catalytic parameters for synthetic substrates and bovine collagen I
altogether according to Scheme I, employing different numbers of
protonating groups, to obtain the whole picture of the proton-linked
modulation of the MMP-8 enzymatic activity.
Such an approach has indicated that five is the minimum number of MMP-8
residues potentially involved in the functional interaction with
natural as well as synthetic substrates to account for the observed pH
dependence of the enzymatic properties of MMP-8; in the following
discussion, we will refer to these five residues as residue i
corresponding to that displaying a pKa1. Therefore,
the application of Scheme I with five protonating groups leads to
fairly complex equations for the analysis of catalytic parameters, such
as,
Fig. 1A shows the
electrophoretic pattern of the bovine collagen I fragmentation by human
neutrophil collagenase MMP-8 at pH 7.0 and 37 °C. It has been
reported by many other authors that proteolysis of collagen is
occurring through a very specific recognition mechanism, which displays
different determinant factors for the various types of collagen (19).
In the case of collagen I this process brings about the cleavage of a
specific peptide bond between Gly775 and the residue in
position 776 (which can be either Leu or Ile) (4, 7), leading to the
formation of 1/4 and 3/4 fragments. This event is also
observed in Fig. 1A, and the two populations are indicated
on the side of the electrophoretic pattern. Obviously, the amount of
the two populations varies as a function of the incubation time of
MMP-8 with bovine collagen I, and the temporal evolution is reported in
Fig. 1B for the processing of 1 µM bovine collagen I with 0.01 nM MMP-8 with both fragments showing a
bell-shaped time behavior of their concentration. Such a feature
suggests the occurrence of a serial proteolytic pathway, which implies the further fragmentation of both the 3/4 and the 1/4 fractions. Furthermore, Fig. 1B shows that the temporal
evolution of the two fragments is not perfectly overlapping, indicating
the occurrence of an intermediate fragmentation state. Therefore, we
have tried to model this process with a minimum reaction scheme (Scheme
II), where A is the whole collagen, B is
the 3/4 fragment, D is the 1/4 fragment, C is the
intermediate fragmentation state (e.g. 1/2 fragment, as in our modeling of Scheme I), and E is the smallest fragmentation state (i.e. 1/n of D, in our case
n = 2). The representation appears adequate to describe
the process, at least to a first approximation, even though other
schemes might likely turn out to be equally good.
Fig. 1C reports the circular dichroism spectrum of the
gel-entrapped bovine collagen I in the UV region between 190 and 240 nm
during the proteolytic processing by MMP-8 under the same experimental conditions employed for the experiment reported in Fig. 1B.
Gel entrapping avoids formation of collagen aggregates and fibrils at a
physiological pH range. The CD spectra so obtained are quite in
agreement with those obtained for collagen-related peptides (24, 25)
showing the characteristic collagen triple-helix maximum in the
223-225 nm range. This result allows us to have a "quasinative"
model for evaluating structural rearrangements during proteolytic
processes on collagen. It appears evident that over the first 20 h, during which the 3/4 and the 1/4 fragments are building up (see Fig. 1B), very minor changes can be
detected for the dichroic spectrum, suggesting that the unwinding of
the molecule is very limited, if any. On the other hand, during the following 30 h a large variation of the spectrum is observed (see Fig. 1C), in particular a great decrease of the peak
intensity at 223-225 (24), which corresponds to a large fraction of
the further fragmentation of the collagen molecule (see Fig.
1B).
As an internal control, acid solution of bovine collagen I and/or
gel-entrapped collagen samples at the pH and temperature condition of
the proteolytic assays were monitored for a time longer than 50 h,
showing no significant changes in CD spectra (data not shown).
Therefore, such observations suggest that the 1/4 and the
3/4 fragments retain most of the secondary structural arrangement of the native collagen, whereas further fragmentation leads
to a significant unwinding of the molecule.
In view of these observations, which indicate that upon the first
cleavage step no major structural change is occurring in the collagen
fragments, the investigation has been primarily focused on the first
step of the proteolytic processing of bovine collagen I by MMP-8,
comparing it to the behavior observed with two synthetic substrates
whose cleavage site closely resembles that of the natural substrate. In
this way, we can observe the role of possible additional regions of the
macromolecular natural substrate on the enzymatic action of the
neutrophil collagenase.
Fig. 2 displays the
pH-dependent behavior of
kcat/Km (A),
kcat (B), and Km
(C) for MMP-8 with two synthetic substrates, namely MCA-1
and MCA-2 (see "Materials and Methods"). The global analysis of
catalytic parameters2 for the
proteolytic processing of these two synthetic substrates indicates that
a satisfactory description of enzymatic properties requires the
involvement of at least three protonating groups to account for the
proton-linked functional modulation. However, it must be outlined that
the different amino acid sequence of the two substrates seems to have
some influence on the absolute values of the catalytic parameters as
well as on the pKa shifts observed upon substrate
binding, thus resulting in a different pH-dependent profile
for the catalysis of the two substrates (see Fig. 2). Thus, whereas
values of pKa for the free enzyme (i.e.
pKU values) are independent on the substrate, as
expected (see Table I), in the
substrate-bound enzyme (i.e. pKL
values) these values strongly depend on the substrate. Such an
observation is very important, because it may represent relevant
information for the final target of mapping the functional role played
by different residues involved in substrate binding and cleavage.
Cleavage of Bovine Collagen I by Neutrophil Collagenase MMP-8
EFFECT OF pH ON THE CATALYTIC PROPERTIES AS COMPARED TO
SYNTHETIC SUBSTRATES*
,,
Department of Experimental Medicine
and Biochemical Sciences, University of Roma Tor Vergata, Via di Tor
Vergata 135, I-00133 Roma, Italy, the § Department of
Molecular, Cellular, and Animal Biology, University of Camerino, Via F. Camerini 2, I-62032 Camerino (MC), Italy, and the ¶ PoliFarma,
Via di Tor Sapienza 138, I-00155 Roma, Italy
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
80 °C until use. The aliquots were then run on a 12% SDS-polyacrylamide gel
electrophoresis, as described previously (23), and the gels were
stained by using Coomassie Blue (see Fig. 1A). The intensity
of different bands was measured with a gel scanner (LKB 2202 UltraScan), and their concentration was determined by comparison with
standard collagen solution.
nor-valine-His-Ala-DPA-NH2 (i.e. MCA-1) and
MCA-Pro-Leu-GlyLeu-DPA-Ala-Arg-NH2 (i.e.
MCA-2); both substrates were a gift of Dr. G. Knight, Strangeways Res. Laboratory, Cambridge, U.K. Experiments were carried out at a final
0.01 nM concentration of purified activated MMP-8 at
37 °C in 50 mM Tris/HCl, 0.1 M NaCl, 10 mM CaCl2 plus 0.05% Brij 35 buffered at
different pH values (between 10.5 and 4.8). The pH value was measured
before and after the experiment, and only measurements in which no pH
change was observed have been taken into account. Experiments at pH
4.8, 5.7, and 6.2 have been also performed in 5 mM MES, 0.1 M NaCl, 10 mM CaCl2, and no
difference for enzymatic activities between two buffer systems
(i.e. MES and Tris/HCl) was observed. The substrate was
diluted in Me2SO, and preliminary experiments at all pH
values investigated demonstrate that the addition of Me2SO
to the incubation mixture does not affect MMP-8 activity. In the case
of synthetic substrates, MCA-1 and MCA-2 assays were carried out by
continuously monitoring the increase in fluorescence at 393 nm after
excitation at 328 nm, using a Jobin-Yvon spectrofluorimeter (model
JY-3), and the amount of substrate catalyzed was calibrated at every
pH, letting the catalysis reaction go to completion and measuring the
amplitude of the signal; only experiments for which a linear dependence on substrate concentration has been observed have been used for the
analysis. The measurement of the initial velocity has been referring to
a time period over which less than 10% of the substrate was degraded
during the assay, and data were normalized and expressed as nanomoles
of cleaved substrate/s. Assays were made with substrate concentrations
between 5 × 10
7 and 104
M, i.e. spanning the range of
Km values, and no absorptive quenching effect has
been detected.
View larger version (13K):
[in a new window]
Scheme I.
where k can be either
kcat/Km or
kcat, Ki (i = 0 ... n) are the protonating equilibrium constants (with
K0 = 1, K = KU in the case of
kcat/Km and K = KL in the case of kcat)
and × = [H+]. Slightly different is the equation
for the proton-linked behavior of obsKm,
which is as follows,
(Eq. 1)
where 0Km is the Michaelis-Menten
equilibrium constant of the unprotonated enzyme, × has the same value
as in Equation 1, KU and
KL are the proton binding equilibrium constants
for residue(s) i to the free enzyme and the substrate-bound
form, respectively.
(Eq. 2)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
A, electrophoretic pattern of the bovine
collagen I (at a concentration of 1 µM) fragmentation at
37 °C by MMP-8 (final concentration 0.01 nM) at
different time intervals (as indicated). The main 3/4 and
1/4 fragments are marked on the side portion of the figure.
B, concentration of 1/4 and 3/4 fragments of
bovine collagen I as a function of time after addition of MMP-8 (0.01 nM final concentration). Experimental values have been
obtained by measuring the optical density of bands reported in
A with a gel scanner. The continuous lines have been
obtained by simply applying the catalytic parameters reported in Table II to obtain k1 in Scheme II and thus the production
of species B and D after the first step (see Scheme II). The values of
k2 (0.05(±0.02) h 1), k3
(0.04(±0.01) h1), and k4 (0.12(±0.03)
h1) have been obtained by least-squares fitting of
experimental data according to a Runge-Kutta algorithm applied to
Scheme II. C, circular dichroism spectra of agarose
gel-entrapped bovine collagen I as a function of time (as indicated)
after exposure to proteolytic fragmentation by MMP-8. For further
details, see text.
View larger version (10K):
[in a new window]
Scheme II.
View larger version (12K):
[in a new window]
Fig. 2.
pH dependence at 37 °C of
kcat/Km
(A), kcat
(B), and Km
(C) for the enzymatic catalysis by MMP-8 of two
synthetic fluorogenic substrates, namely MCA-1 ( 
) and MCA-2
(x). Continuous lines have been obtained by using Equations 1 and
2, employing parameters reported in Table II. For further details, see
text.
Values of pKa in the substrate-free (pKU) and
in the substrate-bound (pKL) form of MMP-8 resulting
from the global fitting of two synthetic substrates (i.e. MCA-1 and
MCA-2) and the natural substrate bovine collagen I
This appears unequivocal in the case of the natural substrate bovine
collagen I (see Fig. 3 and Table I),
where a marked displacement of the optimal overall activity
(i.e. kcat/Km) toward a much more alkaline pH with respect to synthetic substrates is
observed (see Fig. 3A), suggesting that in this case an
important functional role could be played by residues characterized by
fairly high pKa values. A global analysis of the
catalytic behavior of MMP-8 suggests that at least five residues must
be involved to account for the overall functional modulation with the
two synthetic substrates and the bovine collagen. Obviously, these five
residues are not playing the same role for all three synthetic and
natural substrates, but there is some overlapping as well as some
specific behavior for one substrate and not for the other one. The
situation is rendered more cumbersome by the fact that not all residues
modulating one parameter (e.g. Km through a pKa shift upon substrate binding) are affecting
another one (e.g. the catalytic rate constant
kcat) and vice versa.
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In particular, in the case of MCA-1 three residues appear to be
involved in the modulation of Km (see Table I), displaying a pKa shift (i.e.
pKa3 = +0.21, pKa4 = 0.05, and
pKa5 = 0.30); thus, a bell-shaped pH
dependence of Km is observed (see Fig.
2C), because pKa3 brings about an
increase of substrate binding affinity, whereas further
pKa shifts (i.e.
pKa4 and pKa5) induce a
decrease of the substrate association constant. On the other hand,
kcat is mostly affected by
pKL4 (6.71 ± 0.11) and pKL5 (5.85 ± 0.09) and only to a very
marginal extent by pKL3 (7.36 ± 0.09) (see
Table II). Therefore, if we take a
general overview of the modulation mechanism for the enzymatic
catalysis of MCA-1 by MMP-8 we can say that the interaction of MCA-1
(i) does not affect the protonation of the two residues with higher pKa values (i.e. pKa1
and pKa2), (ii) raises the value of
pKa3, bringing about proton uptake by this residue,
but this has a very modest effect on the rate-limiting step kinetic
constant, (iii) decreases significantly the value of
pKa5, and to a lesser extent that of
pKa4, with varying effects on
kcat (see Fig. 2B). In fact,
protonation of residue with pKL4 greatly
increases the catalytic rate-limiting step, whereas the opposite occurs
upon protonation of the group characterized by
pKL5. As a whole, the overall catalytic rate constant kcat/Km for MCA-1
shows an enhancement upon the protonation of residue 3, mostly because
of the increased substrate affinity, which is further accentuated by
the protonation of residue 4, mostly because of the raised
kcat (see Table II). The decrease of both
Km and kcat upon the
protonation of residue 5 leads to a marked decrease of the
kcat/Km (see Fig.
2A).
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Also in the case of MCA-2 there are three protonating groups that
appear to be involved in the modulation of Km
(i.e. pKa1 = +0.05,
pKa3 = 0.16, and
pKa4 = 0.24, see Table I), two of which
(namely pKa3 and pKa4) are the
same involved in the case of MCA-1, and they show a
pKa decrease on MCA-2 binding, bringing about a
decrease of the substrate affinity (corresponding to an increase of
Km, see Fig. 2C and Table I). A third
residue, which displays a higher pKa value
(i.e. pKa1), appears specific to the
interaction of MCA-2 with MMP-8, and its pKa increase upon substrate binding is responsible for the enhancement of
substrate affinity (corresponding to a decrease of
Km) as the pH lowered from 10 to 8 (see Fig.
2C). Furthermore, whereas the protonation of the residue
characterized by pKL1 does not influence the
rate-limiting step, the protonation of the residues characterized by
pKL3 and pKL4 affects
kcat as well (see Table II), bringing about in
both cases a significant rate enhancement (see Fig. 2B). In
addition, a third residue, whose protonation is not influenced by the
substrate interaction (i.e. pKL5),
also affects kcat, inducing a marked reduction
of this rate constant (see Table II). The overall catalytic behavior
for the enzymatic processing of MCA-2 shows a slight increase of
kcat/Km after the protonation
of residue 1, which is completely because of the decrease of
Km; after the protonation of residue 3 we have a
marked rise of kcat/Km,
totally referrable to an increase of kcat,
whereas the protonation of residue 4 keeps kcat/Km essentially unchanged
because of a balancing between an increase of
kcat and a decrease of substrate affinity (corresponding to an increase of Km, see Fig.
2C). Protonation of residue 5 leads to a decrease of
kcat/Km, which mirrors the
behavior of kcat, Km being
unchanged because pKa5 = 0.
As a whole, the catalytic behavior of these two synthetic substrates displays some similarities, but also differences, likely related to the variation of amino acid sequence of the peptide. In particular, for both substrates (i) residue 2 does not appear to play any role, (ii) after protonation of residue 3 the affinity is closely similar, (iii) the highest kcat, which is observed after the protonation of residue 4, has a closely similar value. On the other hand, several differences can be outlined between the two synthetic substrates, so that although binding of MCA-1 brings about a raising of pKa3, in the case of MCA-2 we observe a decrease of pKa3 (see Table I), indicating that the interaction mode with this residue is different for the two substrates; MCA-1 brings about a proton uptake, whereas MCA-2 leads to a proton release. Furthermore, although only the interaction of MCA-1 with MMP-8 induces a specific variation on the protonation properties of residue 5, in the case of MCA-2 we observe a pKa shift of residue 1, which is unchanged upon interaction of MMP-8 with MCA-1.
A different situation can be observed for bovine collagen I, because in
this case at least four residues are modulating Km (see Table I), three of which are overlapping with those involved in
the functional modulation of synthetic substrates (being characterized by pKa1 = 3.66,
pKa3 = 2.05, and
pKa4 = +2.15), whereas the other one is
specific of the interaction of MMP-8 with bovine collagen I
(i.e. pKa2 = 2.79). In
addition, the interaction of bovine collagen I with MMP-8 is characterized by a sequence of protonating groups, which alternatively increase (e.g. pKa1 and
pKa4) and decrease (e.g.
pKa2 and pKa3) the
substrate binding affinity; the resulting pH dependence is then more
complex than for the synthetic substrates (see Fig. 3C).
However, only three of these residues (namely residue 2, residue 3, and
residue 4) seem to be relevant for the modulation of
kcat; the first two bring about a rate
enhancement and the third leads to a dramatic reduction of the kinetic
constant for the rate-limiting step (see Table II). The overall
catalytic behavior displays a steep raising of
kcat/Km at very alkaline pH,
which is wholly attributable to a marked increase of collagen binding
affinity related to the proton uptake of residue 1. Proton release of
residue 2 upon collagen binding brings about a drastic decrease of
substrate binding affinity, which induces a pronounced reduction of
kcat/Km (only partially
compensated by the increase of kcat upon
protonation of this residue in the substrate-bound form, see Table II).
A drastic increase in the catalytic efficiency occurs upon protonation of residue 3 in the collagen-bound form, whose proton release upon
substrate binding is, however, accompanied by a marked decrease of the
affinity for collagen, bringing about a modest but meaningful reduction
of the overall enzymatic activity
kcat/Km (see Fig.
3A). Proton uptake of residue 4 drastically reduces
kcat and
kcat/Km in spite of a
moderate increase of collagen binding affinity (as indicated by the
decrease of Km, see Fig. 3C).
It appears clear as there are different types of groups regulating the enzymatic behavior of MMP-8, and their role has a more or less general relevance. As a matter of fact, residues 3 and 4 seem the most central ones, because their pKa values are always affected by substrate binding, suggesting their involvement in the interaction, and their protonation always influences the rate-limiting step kcat, indicating that they are also involved in the formation of the catalytic intermediate(s) (see Tables I and II). Such a behavior indeed suggests that their location is very close to the active site and/or the primary specificity site(s), and their involvement is occurring for all types of substrates, even though the extent of the effect indeed may depend on the amino acid sequence of the substrate. On the other hand, residue 5, whose protonation in the substrate-bound species is important for the modulation of kcat in all substrates investigated (see Table II), is usually not involved in the substrate interaction, because its pKa does not vary (except for the binding of MCA-1) upon the formation of the ES complex (see Table I). Residue 1 appears involved only in the recognition process (in the case of MCA-2 and bovine collagen I), whereas residue 2 comes into play only for the modulation of the enzymatic activity on bovine collagen I (see Tables I and II). This behavior suggests that residue 5 is crucial for the enzymatic activity, such that its protonation essentially abolishes the function of the enzyme, but it does not modulate the activity. Residue 1 appears to lie not in the immediate proximity of the active site, but its modulatory role appears very much related to the ionic properties of the substrate, because its influence on MCA-2 binding might be connected to the presence of an Arg residue. In the case of residue 2, its behavior suggests that the location is fairly separate from the catalytic site and/or the primary specificity site(s), probably lying outside the interaction surface between MMP-8 and small synthetic substrates.
The identification of the five residues is a very complex task,
but some considerations can be made to identify potential candidates.
In the case of residues 3 and 4, their pKa values
suggest that they could be histidyl residues, possibly His162 (involved in the interaction with synthetic
inhibitors, see Ref. 27), but other candidate(s) might be one (or more)
of the histidyl residue(s) coordinating the catalytic Zn2+
atom. Thus, although other groups might be as well responsible for this
proton-linked modulation, the protonation state of N
or
N
of the imidazole could be important in modulating the reactivity of the Zn2+ atom. In any case, these two
residues, whose pKa are affected by both small
synthetic and large macromolecular substrates (see Tables I and II),
appear to undergo a much larger environmental change upon interaction
with collagen than with synthetic substrates (as indicated by the much
larger pKa shifts, see Table I). It clearly
suggests that the interaction of MMP-8 with bovine collagen I brings
about a very large and widespread induced fit conformational change of
the enzyme, which drastically differs from that occurring upon binding
small synthetic substrates. Therefore, even groups, which are not in
the immediate proximity of the catalytic site, may undergo relevant
variations of their interaction network, and thus of their protonation
properties. This seems to be the case for residues 1 and 2, even though
we have to distinguish their substrate-linked effect. In fact, residue
1 displays only an effect on Km (and thus indirectly
on kcat/Km, but not on
kcat, see Table I), which indicates that this
additional residue is likely coming into play only as a main element of
the interacting surface of MMP-8, being observable only for the
interaction with MCA-2 and with bovine collagen I. This residue takes
up protons upon substrate binding, being always protonated in the
substrate-bound form and likely representing a crucial anchoring
charged residue for the substrate molecule. On the other hand, residue
2 shows an effect on both kcat and
Km but only for bovine collagen I, suggesting that
this is a residue that plays a role also in the correct orientation of
the substrate for its enzymatic processing. This might mean that either
(i) it is a residue far enough from the active site but that exerts a
sufficient constraint on the substrate (when it interacts) to become
crucial in facilitating the correct orientation for the enzymatic
cleavage or (ii) it is a residue that is not influenced by the small
conformational change induced by synthetic substrate(s). This being the
case, the behavior observed indicates that only the gross structural change following the interaction with bovine collagen I alters its
pKa, pushing it to play a role in modulating the catalytic rate-limiting step. In either case, collagen binding induces
a proton release by this group, and the relatively high pKa value in the substrate-free form (see Table I)
suggests it to be either an amino group (such as from a lysyl or
arginyl residue) or a phenolic group from tyrosine. In this respect, it may be tempting to relate it to the residue Tyr189, which
indeed is along the possible cleft where a large linear substrate, like
collagen, would interact with MMP-8 and has been suggested to be a
crucial residue for the collagenase
activity.3 In the case of
residue 5, which displays the lowest pKa values (see
Table I), a potential candidate might be Glu198, which is
in close proximity to the catalytic Zn2+ (27) and whose
protonation could lead to a much less active enzyme, as indeed it is
observed in the case of residue 5 (see Table II). However, it must be
pointed out that the role of Glu198 is still a matter of
debate, because in matrilysin (MMP-7) it has been shown that its
substitution by uncharged residues does not affect the pH dependence of
kcat/Km (26). Therefore, an
additional possibility might be represented by one of the histidyl residues coordinating the catalytic Zn2+ atom and
whose protonation would lead to the cleavage of the Zn2+-imidazole bond and to the complete unactivation of the
enzyme. This being true, it might be possible to envisage the
possibility that the most acid portion of the pH dependence of
catalytic activity in MMP-8 is characterized by a variation of the
coordination state for the catalytic Zn2+ atom.
In conclusion, it appears clear as MMP-8 displays a wide range of
interactions with substrates, this being especially true for large
macromolecular substrates, which involve both residues in the close
proximity to the active site, but also residues quite far apart from
the catalytic cleft. This behavior, which is not unexpected and was
somehow already predicted on the basis of structural modeling (19), has
been characterized here for the first step of the enzymatic processing
of bovine collagen I (leading to the fragmentation into 1/4 and
3/4 species, see Ref. 4), which is closely similar to the
natural substrate of MMP-8. It envisages a very complex interplay among
different portions of the enzyme molecule in the substrate recognition
and/or processing, as also suggested by the processing of collagen by
MMP-1 (17). Therefore, such a investigation indeed may represent a
first basis for the comprehension of molecules, which efficiently
interfere at different levels with the cleavage of physiological
substrates by MMP-8 as well as by other collagenases.
| |
ACKNOWLEDGEMENT |
|---|
We are thankful for the generous gift of bovine collagen by Prof. E. Menegatti, University of Ferrara.
| |
FOOTNOTES |
|---|
* This work was supported by the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) COFIN Grant 9803184222.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: Dept. of
Experimental Medicine and Biochemical Sciences, Bldg. F-Nord, Rm. 65, University of Roma Tor Vergata, Via di Tor Vergata 135, I-00133 Roma,
Italy. Tel.: 39-06-72596365; Fax: 39-06-72596353; E-mail: coletta@seneca.uniroma2.it.
Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M000283200
2 G. F. Fasciglione, S. Marini, S. D'Alessio, V. Politi, and M. Coletta, submitted for publication.
3 H. Nagase, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
MMP, metalloproteinase;
MMP-8, neutrophil collagenase;
MCA, (7-methoxycoumarin-4-yl)acetyl;
DPA, N-3-(2,
4-dinitrophenyl)-L-2,3-diaminopropionyl;
MCA-1, MCA-Pro-cyclohexylalanine-Glynor-valine-His-Ala-DPA-NH2;
MCA-2, MCA-Pro-Leu-GlyLeu-DPA-Ala-Arg-NH2;
MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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