Biology Reference
In-Depth Information
Collagenolytic activity
Full length
(%)
'Cat' only
(%)
Cutter
(%)
Unwinding
(%)
1
MMP-1
1
Zn
2+
100
100
100
100
Y191T
1
MMP-1(Y191T)
1
1
Zn
2+
23
60
27
<10
3
MMP-3
3
3
Zn
2+
0
0
<10
0
T193 N192
3
LC2
1
1
Zn
2+
3
-
-
100
E184 R183
3
LC3
1
1
Zn
2+
13
66
346
450
Cat
Hinge
Hpx
Fig. 5.4 Collagenase, unwinding and “cutter” activities of MMP-1, MMP-3 and MMP-1/MMP-3
chimeras. Schematic representations of MMP-1, MMP-3 and MMP-1/MMP-3 chimeras are shown
on the
left
.
Light grey
represents MMP-1 derived sequence,
dark grey
is sequence originating from
MMP-3. At the fusion point the sequence residues are numbered according to the sequence of
MMP-3 and MMP-1, respectively. The table shows various activities of each construct and values
are in relative activity. All assays were carried out at 25
C. The collagenolytic activity of full-
length MMPs and of the catalytic domain of MMP-1 were measured by quantifying
fragments
generated after SDS/PAGE. The values given are relative activities taking each activity of MMP-1
as 100. The collagenolytic activity of the catalytic domain of MMP-1 is 1/60,000 (0.0017%) of that
of full-length MMP-1. The collagen “unwinding” activity was determined as the ability of each
full-length MMP whose catalytic Glu was mutated to Ala to make triple helical collagen I
susceptible to 3
m
M MMP-1Cat. The raw values obtained as per cent collagen cleaved/h/M
MMP(E200A) mutant were converted to relative values taking the activity of MMP-1(E200A)
as 100. The “cutter” activity is expressed as the ability of the catalytic domain of each MMP (or
MMP variant) to cleave triple helical collagen I in the presence of 2
m
MMMP-1(E200A). The raw
values measured as per cent collagen cleaved/h/M MMPCat was converted to relative values
taking MMP-1Cat as 100
¾
5.5.5 Multiple Steps are Involved in Collagen Unwinding
by Collagenases
The analyses of the unwinding activity of collagen and the ability to hydrolyse
peptide bonds (cutter activity) of MMP-1 and of its mutants have provided
further insights into triple helicase activity (Fig.
5.3
). The collagen unwinding
activity was measured for the ability of catalytically disabled full-length enzyme
(Cat-Link-Hpx) to allow MMP-1Cat to cleave
1(I) chains as described by Chung
et al. (
2004
), and the “cutter” activity as the ability of the Cat domain to cleave
a
1(I)
chains in the presence of full-length MMP-1(E200A). The latter activity was
measured under conditions where the Cat domain alone does not cleave collagen.
Figure
5.3
summarizes the results. In general, when MMP-1 was mutated at a
single site, collagen unwinding activity and collagenolytic activity correlated well,
suggesting that the unwinding activity is a rate-limiting step for collagenolysis.
a
