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activity of the catalytic domain over that of the full-length enzyme should be
similar among different collagenases and their mutants. To address these questions,
we first examined whether the catalytic domain of MMP-1 (MMP-1Cat) alone can
cleave collagen into
and ¼ fragments at 25
C. We found MMP-1Cat could cleave
collagen time and dose dependently, but the activity was only 1/60,000 of that of
full-length MMP-1. A similar minute collagenolytic activity was recently reported
for the catalytic domain of MMP-8 which was roughly 20-fold higher than that of
MMP-1Cat (Salsas-Escat et al.
2010
). When the catalytic domains of various
MMP-1 mutants including MMP-3/MMP-1 chimeras were compared with MMP-
1Cat, there were only small changes in collagenolytic activity, but much larger
reductions with full-length mutants although they were still 12,000-38,000-time
more active than the respective Cat domains (Fig.
5.3
). These results suggest that
the vulnerable state of collagen is present, but it alone is not sufficient for effective
catalysis of triple helical collagen. Non-catalytic domains need to participate in
collagenolysis by making the collagen more susceptible by presumably altering
the triple helical structure. This latter aspect is supported by a study with the
inactive E200A mutant of MMP-1. In the presence of 3
¾
M MMP-1(E200A),
the collagenolytic activity of MMP-1Cat (“cutter” activity) increased about ten-
fold (Chung et al.
2004
). Neither MMP-3 or MMP-3Cat had collagenolytic acti-
vity, but MMP-3Cat cleaved
m
and ¼ in the presence
of MMP-1(E200A). Variable increases in cutter activity of MMP-1 mutants and
MMP-3/MMP-1 chimeras (e.g., a 4.5-fold increase with MMP-1(Y191T) and
a 58-fold increase with LC3 mutant) were also observed in the presence of
MMP-1(E200A) (Fig.
5.3
). These results may be interpreted as indicating that
considerable structural changes are induced in
a
1(I) and
a
2(I) chains into
¾
2(I) chains of collagen
I by interacting with MMP-1(E200A). If MMP-1(E200A) were to simply stabilize
the vulnerable state of collagen without causing structural changes in triple helical
collagen, it would give a similar fold increase in collagenolytic activity among
these mutants as it only affects the concentration of the substrate in a susceptible
state, but not substrate specificity.
a
1(I) and
a
5.5.4 Docking of Collagenase-Cleavage Site of Collagen I
into the Collagenase Active Site
To assess the conformation of the
a
chain that collagenases can accept in the active
site, the
a
2
(I) chain of triple helical collagen I reported by Orgel et al. (
2006
) was
docked into the active site cleft of MMP-1 (see Fig.
5.4
). In this model, the carbonyl
oxygen of Gly
757
of the Gly-Leu bond was directed to the catalytic zinc and the
Leu
756
to the S1
0
pocket, which gives a good fit of the side chain of the P
1
0
Leu to the
S1
0
pocket. However, P
2
(Gln) and P
3
(Pro) are in a different orientation from those
of the peptide substrate modelled onto crystal structures of MMP-8Cat (Grams et al.
1995
). In addition, although
a
2(I) has a slightly open structure around the cleavage
